melissa officinalis extract inhibits herpes simplex virus-i...

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iMedPub Journals http://www.imedpub.com 2016 Vol. 2 No. 2: 8 Research Article Herbal Medicine: Open Access ISSN 2472-0151 1 © Under License of Creative Commons Attribution 3.0 License | This article is available in: http://herbal-medicine.imedpub.com/archive.php DOI: 10.21767/2472-0151.100014 Karen Denzler L 1 , Trung Huynh P 1 , Bertram Jacobs L 1 and Jeffrey Langland O 1,2 1 Biodesign Instute, Arizona State University, Tempe, Arizona 85287-5401, USA 2 Southwest College of Naturopathic Medicine, Research Department, Tempe, AZ 85282, USA Corresponding author: Jeffrey Langland O [email protected] Southwest College of Naturopathic medicine Research Department Tempe, Arizona 85252, USA. Melissa officinalis Extract Inhibits Herpes Simplex Virus-I Glycoprotein B Interacon with Heparin Sulfate Abstract Herpes simplex virus 1, HSV1, is the primary cause of herpes labialis in humans. Drugs like acyclovir and its derivaves are available for treatment, but with increased use and the number of immune compromised paents, the development of resistance to these drugs is increasing. Extracts of the botanical, Melissa officinalis, have previously been reported to contain anviral acvity toward HSV1. Our inial studies confirmed earlier results that constuents of Melissa officinalis interacted directly with the virus and inhibited HSV1 binding to cells during the iniaon of infecon. Further studies demonstrated that a component in Melissa officinalis bound specifically to the viral glycoprotein B. Virion structure was shown to be stable at low concentraons of Melissa officinalis, however at a ten-fold higher concentraon than that which inhibited binding, virion structure was completely disrupted suggesng a second, virucidal, mode of inhibion. Melissa officinalis was shown to inhibit other alpha herpes viruses as well as having intermediate inhibitory acvity against other viruses from the adenoviridae, poxviridae, papovaviridae, and rhabdoviridae families. Keywords: Herpes simplex; Melissa; Lemon balm; Botanical; Heparin sulfate Received: April 14, 2016; Accepted: April 25, 2016; Published: April 30, 2016 Introducon Herpes simplex virus type 1 (HSV1) is a member of the Herpesviridae family that primarily causes herpes labialis (cold sores) in humans. HSV1 also causes facial, pharyngeal, ocular, central nervous system infecons, and in rare cases, encephalis. It is seroprevalent in almost 58 percent of the US populaon and increases in prevalence with age [1,2]. Worldwide an esmated 60-95 percent of adults are infected with HSV1 [3]. Although HSV1 infecon occurs predominantly at oral sites, an increase in infecon at genital sites has been reported [1]. HSV1 infecon is transmied through mucocutaneous sites via direct contact with a lesion or with body fluids like saliva, respiratory droplets, or genital secreons [3,4]. Primary infecon occurs in epithelial cells of the oral mucosa or lips and can be either asymptomac or cause erythymatous vesicles in the dermis and epidermis which is followed by establishment of latency in the innervang sensory neurons [5-9]. HSV1 is transported to the cell bodies of the neuron where latency is established, oſten in the trigeminal or dorsal root ganglia [4,10]. During latency, the viral lyc genes are repressed and the viral genomes are maintained as circular, extrachromosomal episomes. Periodically, the viral genomes in some of the neurons reacvate and virus travels down the axon to the original site of infecon where it can cause recurrent disease resulng in skin ulceraons and pain [4,11]. Current treatment toward HSV1 infecon typically uses acyclovir and the derivaves, famciclovir and valacyclovir [1,12]. Acyclovir is a guanine nucleoside analog that is converted by the viral thymidine kinase to acyclovir monophosphate. Cellular kinases add two more phosphate groups to generate acyclovir triphosphate that compevely inhibits the viral DNA polymerase and is incorporated into the viral DNA during genome replicaon resulng in chain terminaon [12]. The effecveness of these drugs can be limited in paents with compromised immune systems or in those experiencing chronic HSV infecons. In these cases, the potenal for the virus developing drug resistance is higher [13,14]. In addion, these drugs can have side effects that include nausea, diarrhea, and voming. Drugs like foscarnet, a pyrophosphate analog that binds the viral DNA polymerase, and cidofovir, a nucleode analog that does not require phosphorylaon by the viral thymidine kinase, can be used when acyclovir resistance has occurred, although these drugs display reduced bioavailability or nephrotoxicity, respecvely [15-18].

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Page 1: Melissa officinalis Extract Inhibits Herpes Simplex Virus-I …herbal-medicine.imedpub.com/melissa-officinalis-extract... · 2017-01-06 · Melissa officinalis Extract Inhibits Herpes

iMedPub Journalshttp://www.imedpub.com

2016Vol. 2 No. 2: 8

Research Article

Herbal Medicine: Open AccessISSN 2472-0151

1© Under License of Creative Commons Attribution 3.0 License | This article is available in: http://herbal-medicine.imedpub.com/archive.php

DOI: 10.21767/2472-0151.100014

Karen Denzler L1,Trung Huynh P1,Bertram Jacobs L1 and Jeffrey Langland O1,2

1 BiodesignInstitute,ArizonaStateUniversity,Tempe,Arizona85287-5401,USA

2 SouthwestCollegeofNaturopathicMedicine,ResearchDepartment,Tempe,AZ85282,USA

Corresponding author: JeffreyLanglandO

[email protected]

SouthwestCollegeofNaturopathicmedicineResearchDepartmentTempe,Arizona85252,USA.

Melissa officinalis Extract Inhibits Herpes Simplex Virus-I Glycoprotein B Interaction

with Heparin Sulfate

AbstractHerpessimplexvirus1,HSV1,istheprimarycauseofherpeslabialisinhumans.Drugslikeacycloviranditsderivativesareavailablefortreatment,butwithincreaseduse and the number of immune compromised patients, the development ofresistancetothesedrugsisincreasing.Extractsofthebotanical,Melissa officinalis,havepreviouslybeenreportedtocontainantiviralactivitytowardHSV1.OurinitialstudiesconfirmedearlierresultsthatconstituentsofMelissa officinalisinteracteddirectlywiththevirusandinhibitedHSV1bindingtocellsduringtheinitiationofinfection.Further studiesdemonstrated thatacomponent inMelissa officinalis boundspecifically to theviralglycoproteinB.Virionstructurewasshowntobestableat lowconcentrationsofMelissa officinalis,howeverata ten-foldhigherconcentrationthanthatwhichinhibitedbinding,virionstructurewascompletelydisrupted suggesting a second, virucidal,mode of inhibition.Melissa officinalis wasshownto inhibitotheralphaherpesvirusesaswellashaving intermediateinhibitory activity against other viruses from the adenoviridae, poxviridae,papovaviridae,andrhabdoviridaefamilies.

Keywords:Herpessimplex;Melissa;Lemonbalm;Botanical;Heparinsulfate

Received: April14,2016; Accepted: April25,2016;Published: April30,2016

Introduction Herpes simplex virus type 1 (HSV1) is a member of theHerpesviridae family that primarily causes herpes labialis (coldsores) in humans. HSV1 also causes facial, pharyngeal, ocular,centralnervoussysteminfections,andinrarecases,encephalitis.Itisseroprevalentinalmost58percentoftheUSpopulationandincreasesinprevalencewithage[1,2].Worldwideanestimated60-95 percent of adults are infected with HSV1 [3]. AlthoughHSV1infectionoccurspredominantlyatoralsites,anincreaseininfectionatgenitalsiteshasbeenreported[1].

HSV1 infection is transmitted through mucocutaneous sitesvia direct contact with a lesion or with body fluids like saliva,respiratorydroplets,orgenitalsecretions[3,4].Primaryinfectionoccursinepithelialcellsoftheoralmucosaorlipsandcanbeeitherasymptomaticorcauseerythymatousvesiclesinthedermisandepidermiswhich is followedbyestablishmentof latency in theinnervatingsensoryneurons[5-9].HSV1istransportedtothecellbodiesoftheneuronwhere latency isestablished,often inthetrigeminalordorsalrootganglia[4,10].Duringlatency,thevirallyticgenesarerepressedandtheviralgenomesaremaintained

as circular, extrachromosomal episomes. Periodically, the viralgenomes in some of the neurons reactivate and virus travelsdowntheaxontotheoriginalsiteofinfectionwhereitcancauserecurrentdiseaseresultinginskinulcerationsandpain[4,11].

Current treatment toward HSV1 infection typically usesacyclovirandthederivatives,famciclovirandvalacyclovir[1,12].Acyclovir is a guanine nucleoside analog that is converted bytheviralthymidinekinasetoacyclovirmonophosphate.Cellularkinases add twomore phosphate groups to generate acyclovirtriphosphatethatcompetitivelyinhibitstheviralDNApolymeraseandisincorporatedintotheviralDNAduringgenomereplicationresulting in chain termination [12]. The effectiveness of thesedrugs can be limited in patients with compromised immunesystemsorinthoseexperiencingchronicHSVinfections.Inthesecases, the potential for the virus developing drug resistance ishigher[13,14].Inaddition,thesedrugscanhavesideeffectsthatincludenausea, diarrhea, and vomiting.Drugs like foscarnet, apyrophosphate analog that binds the viral DNA polymerase,and cidofovir, a nucleotide analog that does not requirephosphorylationbytheviralthymidinekinase,canbeusedwhenacyclovir resistancehasoccurred, although thesedrugsdisplayreducedbioavailabilityornephrotoxicity,respectively[15-18].

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For the past half a century, the US population has beenincreasingly seeking alternative therapies to chronic diseases[19-21].Astudy in2002estimated19percentofadultsusedabotanical therapy to treat illnesses or conditions [22]. Giventhat herpes virus infection results in lifelong persistence andis associated with recrudescence in many patients, a numberof plant extracts and oils have been analyzed for therapeuticpotentialandincludemembersoftheLamiaceaefamily: lemonbalm,peppermint,prunella,rosemary,sage,andthyme;aswellasbirchbark,Gynura,Manuka,teatree,eucalyptus,ginger,thyme,hyssop,sandalwood,andpropolis[23-32].Thesebotanicalmayofferalternativetherapiesaswellasthepotentialforisolationofnovel,efficaciousactivecompounds.

Melissa officinalis,alsoknownaslemonbalm,hasbeenpreparedas both aqueous extracts or essential oils and tested forantiherpesactivityin vitro[23,33,34].EithertypeofpreparationdisplaysinhibitoryactivitytowardHSV1andanaqueousextractwasshownto inhibitacyclovir-resistantstrainsofHSV1[34]. Inaddition, theuseofa topicalcreammade from lemonbalm inthetreatmentofherpesinfectionoftheskinshowsasignificantreductioninthesizeoflesionsbyday2oftreatment[35].

InfectionbyHSV1 ismediatedbybinding to specificmoleculeson cells using a number of virus-encoded glycoproteins thatare present in the virus envelope. Initial binding occurs withglycoprotein C (gC) and glycoprotein B (gB) which bind to thecellsurfaceglycosaminoglycans,heparansulfateandchondroitinsulfate, or by the recently described interaction between gCand the scavenger receptor, MARCO [36-39]. Glycoprotein D(gD)thenbindstoentryreceptors,nectin-1,nectin-2,HVEM,or3-O-sulphatedheparansulfateandundergoesaconformationalchange that facilitates interactions with viral glycoproteins gB,gH,andgL[40-44].Thefourglycoproteins,gD,gB,gH,andgL,arerequired formembrane fusion and releaseof the viral particleinto the cytoplasm at the external plasmamembrane or fromendocyticvesicles[45,46].

AqueousextractsofMelissa officinalishavebeenshowntoinhibitbindingofHSV1virions to thecell [23,34].Given thehistoricaluseandgrowingbodyofevidence,thisstudysoughttofurtherexamine the antiviral activity of Melissa officinalis towardsHSV1tobetterdelineatethemechanismofactionofthewholebotanicalextract.

Materials and MethodsCell lines and virus stocks Verocells(ATCC)weremaintainedwithMinimalEssentialMedia(MEM, Cellgro) supplemented with 100 IU penicillin/ml, 100µg streptomycin/ml, 2.5 µg amphotericin B/ml, and 10%heat-inactivated fetal bovine serum (HI-FBS, Hyclone). Cells wereincubated at 37°C, 5% CO2 in a humidified chamber. L, Gro2C,and sog9 cell lines weremaintainedwith Dulbecco’sModifiedEssentialMedia(D-MEM)supplementedwith1µg/mlceftriaxoneand10%HI-FBS.

HSV1KOS(akindgiftfromDavidBloom,Univ.ofFloridaCollegeof Medicine), HSV1-1(KOS)∆gC2-3 (a kind gift from RobertVisalli,IndianaUniversityPurdueUniversity),GHSV-UL46(ATCC)

expressingGFP linkedto thetegumentprotein,VP11/12,HSV2(ATCC),andEHV(ATCC)werepropagatedinVerocells.AtfullCPE,cellswerepelleted, resuspended inMEM,2%FBS, and freeze-thawed three times followed by sonication for 2minutes. Celldebris was removed by centrifugation, and viral stocks werealiquoted and stored at -80°C. For some experiments, HSV1KOSandGHSVwerepurifiedusinganIodixanolstepgradientbycentrifugationat140,000xgfor3hoursinanSW28followedbyremovalofthevirusbandlocatedatthe20%to30%interface.ThevirusbandwasdilutedinMEM,2%FBSandpelletedthrougha20% Iodixanolpad.Theviruspelletwas thenresuspended inMEM, 2% FBS, and stored at -80°C. Virus stocks were titeredon Vero cells in the presence of 0.3% human gamma globulin(Sigma),andplaqueswerevisualizedthreedayslaterbystainingwith0.1%crystalvioletin20%ethanol.

ExtractsMelissa officinalis extract was obtained from Vital ForceNaturopathic Compounding (Phoenix, AZ). Briefly, verified anddriedMelissa officinalisleavesweregroundtoafinepowder.Thepowderwasresuspendedata1:8ratio(solid:75%glycerin)andincubated for 2 days at room temperature. The bulk botanicalmaterial thenwasremovedbycentrifugationat3,000xgfor15minutes and the supernatant filtered through a 0.2 µm filter.Thefinalextractwasstoredatroomtemperature.Forreferenceand standardization, all extractionswere consistently found tocontainconcentrationsofnon-volatile solutes rangingbetween25-30mg/mlextract.

Cell viabilityVero cells were incubated with increasing concentrations ofMelissa officinalis extract or vehicle for 24 hours. Cells wereharvested by incubation with 0.25% trypsin for 5 minutesfollowedbygentle resuspension.Cellswerediluted inPBSandmixed 1:1with 0.4% trypan blue (Invitrogen). Percent viabilitywas determined by counting clear versus blue cells using anInvitrogenCountess.

Plaque reduction assays and single cycle growth curvesPlaquereductionassayswereperformedbydilutingvirusstocksand preincubating 100-200 plaque-forming units (pfu) withincreasingconcentrationsofMelissa officinalisextractorvehicle(75% glycerol) for 20minutes.Monolayerswere infected for 1hourat37°CfollowedbyincubationinmediacontainingMelissa officinalisorvehiclefor3daysat37°C.Plaqueswerevisualizedbystainingwith0.1%crystalvioletin20%ethanol.

SinglecyclegrowthcurveswereperformedbyinfectingVerocellsinduplicatewithamultiplicityofinfection(MOI)of5withHSV1KOS. Increasing concentrations ofMelissa officinalis or vehiclewere incubatedwithvirusfor20minutesfollowedby infectionfor1hourat37°C.InfectedcellswerewashedtwicewithwarmPBS,givenfreshmediacontainingMelissa officinalisorvehicle,andwereharvestedat12hourintervals.Infectedcellsplusmediawerethenpelletedat12,000×gfor30minutes,resuspendedinMEM,2%FBS,andstoredat-80°C.Sampleswerefreeze-thawedthreetimes,sonicatedfor2minutes,andtiteredbyplaqueassay.

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Binding/Uptake assaysBindingofHSV1toVerocellswasassayedbyadding100-200pfuHSV1KOS to increasing concentrations ofMelissa officinalis orvehicle 20minutes prior to infection. Viruswas added to pre-chilledVerocellmonolayersandincubatedfor2hoursat4°Ctoallowbinding.CellswerewashedtwotimeswithPBStoremoveunboundvirus,completemediaadded,andthecells incubatedfor3daysat37oCfollowedbycrystalvioletstainingtovisualizeplaqueformation.

Uptake of HSV1 into Vero cells was assayed by infecting pre-chilled Vero cellmonolayerswith 100-200 pfuHSV1 KOS for 2hoursat4°C in completemedia (withoutMelissa officinalis) toallow for cell binding. Monolayers were washed with PBS toremoveunboundvirus,mediawasthenaddedwhichcontainedincreasing concentrations ofMelissa officinalis or vehicle, andincubatedat37°Cfor3daysfollowedbycrystalvioletstainingtovisualizeplaqueformation.

Western blotsHSV1KOSwasusedtoinfectVerocellsatanMOIof5.Melissa officinalis was added to virus at various times prior toinfection,atthetimeofinfection,oratvarioustimesfollowinginfection.Monolayerswerefedwith3mlsofcompletemediaor media containing Melissa officinalis or vehicle after 1hourofincubationat37°C.Following18hoursofincubation,cellswerewashedtwotimeswithPBSandlysedwith1XSDSsample buffer (125mM Tris-Cl, pH 6.8, 25% glycerol, 2.5%SDS,100mMβ-mercaptoethanol,0.025%bromophenolblue,10%Protease inhibitorcocktail (ThermoScientific)).Sampleswere separated on 10% polyacrylamide gels, transferred toPVDF membrane in blotting transfer buffer (10mM CAPS,20% methanol), and blocked in blotto (25mM Tris, pH 7.5,137mMNaCl,2.5mMKCl,0.025%Tween,3%powderedmilk).MousemonoclonalantibodiestoICP4(akindgiftfromDavidBloom,Univ.ofFloridaCollegeofMedicine), ICP8,gC,VP16,GAPDH (Abcam), and gD (Virusys) were diluted accordingto manufacturer’s specifications. Detection was performedusing secondary goat anti-mouse IgGor goat anti-rabbit IgGconjugated to horseradish peroxidase (Santa Cruz) in thepresenceofachemiluminescentsubstrate(Biorad).

ImmunofluorescenceIodixanol-purifiedGHSV-UL46wasusedtoinfectVerocellsseededat25%confluencyovernightonaglasscoverslipatanMOIof50.VariousconcentrationsofMelissa officinalisorvehiclewereaddedtovirus20minpriortoinfection,andcellswereincubatedat 4°C for 2 hours to allow virus binding. Cells were washedwith PBS, fixed in 4% formaldehyde (Thermo Scientific), andquenchedin50mMNH4Cl.Cellmembranewasstainedwith5µg/mlWheatGermAgglutinin(WGA,MolecularProbes)andnucleiwerestainedwith4’,6-Diamidino-2-phenylindoledihydrochloride(DAPI,Sigma).CoverslipsweremountedontoglassslideswithProlongAntifadeReagent(LifeTechnologies)andvisualizedwithaLeicaTCSSP8ConfocalLaserScanningMicroscopeusinga40Xlens (performed at Veteran’s Administration Hospital ResearchCenter,Phoenix,Arizona).

Heparin-agarose binding assaysHeparinconjugated toagarose (Sigma)was incubatedwith1×106pfuHSV1KOSintheabsenceofcompetitororinthepresenceofvariousconcentrationsofMelissa officinalisextractfor1hourat 4°C. Heparin-agarose was washed and pelleted two timesfollowedbytheadditionof1×SDSsamplebuffer.Sampleswereanalyzed forHSV1bindingbyWesternblot for thepresenceofHSV1 gB. Control samples included non-conjugated agarosealoneand theadditionofvehicle inplaceofMelissa officinalis (datanotshown).

Virion stability assayA20%sucrosepadwas loadedwith1×106pfuHSV1KOSthathad been untreated or treated with increasing concentrationsofMelissa officinalis,vehicle,or0.5%Triton×-100for1hourat37°C.Sampleswerecentrifugedat140,000xgfor80minutesinanSW55rotor,andpelletswereresuspendedin1×SDSsamplebuffer followedbyWesternblotanalysis for tegumentprotein,VP16,andoutermembraneprotein,gD.

Depletion assayHis-taggedHSV1 glycoproteins from the virulentMcKrae strainwereexpressedfromplasmids,pPEP98(gB)andpPEP99(gD)(akind gift fromVladimir Chouljenko, Louisiana StateUniversity).VerocellsweretransfectedwitheachplasmidusingLipofectamine2000. Forty-eight hours post-transfection, cells were lysed inNP-40 lysis buffer. The his-tagged glycoproteins B and D weresubsequentlypurifiedusingananti-6XHisantibodyconjugatedagarose resin. The binding of the gB and gD to the resin wasverifiedbyWesternblotanalysis.Melissa officinalisextract(100µl)wasincubatedwiththeresinaloneorwithresinboundwithglycoproteinBorD.Following30minincubationat4°C,theresinwas pelleted at low speed, and the supernatant removed andassayedforHSV1inhibitoryactivityinaplaqueassay.

StatisticsStatisticalanalysiswasperformedusingPrism6 forMACOSX,GraphPad Software, Inc. For immunofluorescence, a one wayANOVAfollowedbyaTurkeytestformultiplecomparisonswasperformedtodeterminedifferencesbetweengroups.

ResultsExtracts of Melissa officinalis inhibit HSV1 cytopathic effect and plaque formationExtracts fromtheplant,Melissa officinalis,havebeenreportedto affect replication of HSV1 while having low toxicity to cellmonolayers[23,34].Theseresultswereconfirmedbyincubatingincreasing concentrations of Melissa officinalis extract withVero cell monolayers alone and by incubating increasingconcentrations of Melissa officinalis or vehicle with Vero cellsinfected with HSV1. Figure 1A shows that concentrations ofMelissa officinalisupto18µl/mlofextracthadnomicroscopicallyvisibleeffectsongrowthormorphologyoftheVerocells.WhencellswereinfectedwithHSV1,typicalcytopathiceffect(CPE)duetoviral infectionwasseen inuntreatedcells (Figure 1B).Upon

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theadditionofMelissa officinalisduringHSV1infection,CPEwasstill observed with 0.1 µl/ml extract, but was greatly reducedat0.3µl/ml,andcompletely inhibitedat1-3µl/mlextract.ThesameconcentrationsofvehicledidnotinhibitCPEduetoHSV1infection(Figure 1B).

The selectivity index (SI) for Melissa officinalis extract wascalculatedbyincubatingHSV1-infectedVerocellswithincreasingconcentrations of Melissa officinalis and by incubating Verocells with increasing concentrations of Melissa officinalis alone. The EC50, or effective concentration at which 50% ofHSV1 plaque formation was inhibited by Melissa officinalis,was determined to be 0.58 µl/ml extract (Figure 2). The CC50,orcytotoxicconcentrationatwhich50%ofthecellswerenon-viablefollowingtreatmentwithMelissa officinaliswas190µl/ml(Figure 2).ThisgaveanSIofMelissa officinalisextract(CC50/EC50)of327.Notably,thecellviabilitycurveandCC50withvehiclealone(75%glycerin)wasverysimilartothatfollowingtreatmentwithMelissa officinalis (Figure 2).Thissuggests thatthecell toxicityof theMelissa officinalis extractwas likely associatedwith theextraction solution (75% glycerol) and that the SI of Melissa officinalismaybehigherthan327.

Melissa officinalis decreases infective titers of HSV1 in a single cycle growth assayVero cells were infected with HSV1 and treated with 0.3 or 1µl/mlMelissa officinalis or vehicle.At 1, 12 and24hourspostinfection, viruswasharvestedandtitered.Figure 3 shows thatthe titer of untreated HSV1 increased by nearly 3-logs at 24hours post infection (hpi) as compared to the input virus titerat1hpi.HSV1treatedwitheither0.3or1µl/mlvehiclehadasimilarpatternofgrowthincellsasuntreatedvirus.TheadditionofMelissa officinalis at0.3and1µl/mlprior toHSV1 infectionresulted in a rapid 1- and 2-log decrease in titer, respectively,

asmeasuredat1hpi.Melissa officinalis treatmentat0.3µλ/mlresultedinnoadditionalchangeintiterat12hpi,butby24hpi,viraltitersincreasedby3-logstoalevelapproximately1-loglessthanuntreatedvirus(Figure 3). Melissa officinalis treatmentat1µl/mlresultedina1.5-logdecreaseinmeasurabletitersat12hpi,butby24hpia2.5-logincreaseinvirustiterascomparedtoinputtiterwasmeasured.Thisfinaltiterat24hpiwasapproximately2-logs less than untreated virus (Figure 3). Therefore, initialtreatmentwithMelissa officinalis resultedinarapiddecreaseininfectivetiters,butallinfectionswereabletopartiallyrecoverby24hpi.

Melissa officinalis inhibited HSV1 when added prior to infectionBasedontheresultsfromFigure 2, Melissa officinalis appearedtolikelyinhibitHSV1duringtheinitialstepsofinfection.Tofurtherevaluate this, Melissa officinalis was incubated with HSV1 forvarioustimespriortoinfection,atthesametimeofinfection,oratvarioustimespostinfectionfollowedbyanalysisofviralproteinexpression. HSV1 treated with 1 µl/ml Melissa officinalis wascomparedtountreatedorvehicle-treatedHSV1bymeasuringtheaccumulationofimmediateearly(IE),early(E),andlate(L)viralproteins. Figure 4A shows that untreated and vehicle-treatedHSV1 resulted in similar expression of ICP4 (IE), ICP8 (E), andgC(L)proteinsat18hpi.Alternately,HSV1virionstreatedwithMelissa officinalis for60or30minutespriortoinfectionoraddedat thesametimeof infectionresulted incomplete lossofviralproteinexpression(Figure 4A).TreatmentofHSV1-infectedcells30minutespost-infectionresultedinaslightdecreaseinproteinexpression for all viral proteins whereas treatment 60 or 120minutespost infectiondidnotsignificantlyreducethe levelsofIE,E,andLproteins(Figure 4A).

A similar experiment was performed that measured viral

Figure 1 Melissa officinalis inhibits HSV1-induced CPE. (A) Vero cell monolayers were treated with increasing concentrations ofMelissa

officinalis andphotographed24hourslater.(B)VerocellmonolayerswereinfectedwithHSV1ataMOI=5,treatedwithincreasingconcentrationsofMelissa officinalis orvehicle,andphotographedat24hpi.

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replication following incubation withMelissa officinalis. Again,1 µl/ml Melissa officinalis was incubated with HSV1 for 30minutesprior to infection, at the sametimeof infection,or atvarioustimespost infectionandanalyzedfortheproductionofinfectiousvirionsat24hpi.At1hpi theamountof input viruswas measured, and at 24 hpi a 4.5-log increase in titer wasobservedinuntreatedsamples (Figure 4B). However,whenviruswas pretreatedwithMelissa officinalis for 30min or added atthe same time as infection, no HSV1 was detected at 24 hpi.WhenMelissa officinaliswasadded30minpostinfection,a1-logdecreaseinyieldascomparedtoinputtiterwasobserved.Thisagrees with results seenwith protein expressionwhich showsthatEI,E,andLproteinproductionisdecreasedascomparedtountreatedvirus.Similarly,theadditionofMelissa officinalisat1,2,4,and6hourspostinfectionresultedinapproximately3-,2-,1-,and1-logreductionsinyield,respectively.

Melissa officinalis at low concentrations inhibits HSV1 virions from binding to the cellThe results of treatment of HSV1 in the previous single cyclegrowth and protein synthesis assays suggest that Melissa officinalis hasaneffectpriortoorduringbindingoftheviriontothecell.AvirusbindingassaywasperformedbyaddingincreasingconcentrationsofMelissa officinalis toHSV1,incubatingtheviruswith cells at 4oC to allow for adsorption, followed bywashingawayunboundvirusandbotanicalextract,andthenincubatingat37°Ctoallowanyboundvirustoreplicateandformplaques.Figure 5A shows thataconcentrationof0.3µl/mland1µl/mlwasabletoinhibit60%and95%ofthevirusfrombindingtocells,respectively,whereasconcentrationsofvehicleupto3µl/mlhadnoeffectonbindingandplaqueformation.

In addition, a virus uptake assaywas performed by incubatingHSV1 with cells at 4°C to initially allow virus to bind to the

cell followed by the addition of media containing increasingconcentrationsofMelissa officinalisorvehiclewithincubationat37°C.Figure 5Bshowsthat0.3µl/mland1µl/mlhadnoeffectonuptakeofthevirus,whereasahigherconcentrationof3µl/mlinhibiteduptakeof70%ofthevirusintothecells.Vehiclehadnoeffectonuptakeandplaqueformation.

Next,assaysweredonetotestiftheeffectofMelissa officinalis wasspecificforthevirusandorthecells.Figure 5Cshowsthatwhencellswerepre-incubatedwithincreasingconcentrationsofMelissa officinalis followedbywashingand then infectingwithHSV1,concentrationsupto3µl/mldidnotaffectHSV1infectivityorreplication.However,whenvariousconcentrationsofMelissa officinalis were pre-incubated with HSV1 virions followed bypelletingofthevirus,resuspendinginfreshmedia,andinfectingcells,adosedependentresponsewasobservedwhere0.3and1µl/mlinhibitedplaqueformationby42%and92%,respectively.

These results together suggest that Melissa officinalis componentsmay be interactingwith the freeHSV1 virion andinhibitingviralattachmenttothecell.HSV1haspreviouslybeenshowntobindtoheparinin vitroasasurrogatemoleculetotheheparansulfatemoietiespresentonthecellsurface[47].Asanin vitro assay, HSV1 virions (1 × 106 pfu) were incubatedwithheparin-agaroseeitheraloneorwithincreasingconcentrationsofMelissa officinalis.Theagarosebeadswerewashedandboundvirus eluted by boiling in SDS-sample buffer. Western blotanalysisof theelutedmaterial (testing forHSV1gB) showedthat 0.3, 1, and 3 µl/mlwas able to effectively reduced theamount of gB present on the resin by 79%, 96% and 100%,respectively (Figure 5D). HSV1didnotbindtoagarosealoneandincubationwithvehiclehadnoeffectonvirionbindingtotheheparin-agarose(datanotshown).

TheinhibitionofvirionbindingtothecellbyMelissa officinalis

Figure 2 SelectivityindexofMelissa officinalis.AplaquereductionassaywasperformedonVerocellmonolayersinfectedwith100-200pfuofHSV1andtreatedwithincreasingconcentrationsofMelissa officinalis todeterminetheEC50.VerocellmonolayersweretreatedwithincreasingconcentrationsofMelissa officinalis,andtrypanbluestainingwasusedtodeterminecellviabilityandtheCC50.ThecellviabilityofMelissa officinalis-treatedcellswascomparedtovehicle(glycerin)-treatedcells.

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Figure 3 Melissa officinalis decreasesHSV1titers.Verocellswere infectedwithHSV1ataMOI=5andwereeither leftuntreated,orweretreatedwith0.3µl/mlor1µl/mlvehicleorMelissa officinalis.Monolayerswereharvestedattheindicatedtimepointsandtiteredforplaqueformingunits.

Figure 4 Melissa officinalis inhibitsHSV1duringpretreatmentoratthetimeofinfection.(A)HSV1wasleftuntreated,treatedwithvehicle,ortreatedwith1µl/mlMelissa officinalis atvarioustimespriortoinfection,atthetimeofinfection,oratvarioustimespostinfection.At18hpicellswereharvested,andWesternblotanalysiswasperformedtodetectIE(ICP4),E(ICP8),andL(gC)viralproteins.AllsampleswerenormalizedtoGAPDH. (B)HSV1was leftuntreatedorwas treatedwith1µl/mlMelissa officinalis 30minprior toinfection,atthetimeof infection,oratvarioustimepost infection.At24hpimonolayerswereharvestedandtiteredforplaqueformingunits.Inputvirus(at1hpi)hadatiterof5×103pfu/ml(notshown).

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was confirmed by performing a binding assay using HSV1expressingaGFP-labeledtegumentprotein(GHSV).Figure 6A showsfullcolorimagesandgray-scaleimagesofGFPforeachfield (representative image from z-stack). In mock-infectedVero cells, only the cell membrane (stained withWGA) andnucleus (stained with DAPI) could be observed (Figure 6A, panels A and E).IncellsinfectedwithGHSV,GFP-fluorescentvirionsbindingtothesurfaceofthecellswereclearlyobserved(Figure 6A, panels B and F).When cells were infectedwithGHSVinthepresenceof3µl/mlvehicle,aslightreductioninvirionparticlesboundtothecellwasobserved,howeverthisdidnotshowstatisticalsignificance(Figure 6A, panels C and G and Figure 6B).WhencellswereinfectedwithGHSVinthepresence of 0.3 µl/ml Melissa officinalis, an 88% reductioninthenumberofviralparticlesboundpercellwasobserved(Figure 6A, panels D and H and Figure 6B), while 3 µl/mlMelissa officinalis resultedinacompletelossofvirionbinding(Figure 6B).

Melissa officinalis disrupts virion structure only at high concentrationsIt is possible that Melissa officinalis reduces virus binding tothe cell by either blocking virus interactionwith cell receptorsorbydisruptingvirionstructure.Totestforvirusstability,HSV1virionswere treatedwith increasing concentrations ofMelissa officinalisorvehiclefollowedbypelletingthroughasucrosepad.Concentrations of vehicle up to 3 µl/ml did not affect stabilityof thevirionsas seenby thepresenceof theenvelopegDandinternal tegument protein (VP16) in the sucrose pellet (Figure 7). Concentrations ofMelissa officinalis up to 1 µl/ml also didnotaffectvirionstability;howeverMelissa officinalisat3µl/mlcauseddisruptionofthevirionsresultinginlossofenvelopeandtegumentproteins (Figure 7). TreatmentwithTritonX-100was

usedasacontroltoshowcompletedisruptionofvirions.TheseresultsmaysuggestthatMelissa officinalismaybindtothevirusandblockcellattachmentatlowconcentrations(1µl/mlorless),whereas higher concentrations (3 µl/ml or more) may affectvirionstability.

Glycoprotein B binds to the antiviral component(s) present in Melissa officinalis TheHSV1glycoproteinsareinvolvedinbindinganduptakeintothecell.InitialbindinginvolvesgBandgCandthereforerepresentlikely targets for the antiviral activity associated with Melissa officinalis. In order to test the role of gC, anHSV1deleted forthegCgene(HSV1-1(KOS)∆gC2-3)wasusedtotestfortheabilityof Melissa officinalis to inhibit viral replication. HSV1 deletedforgCisaweakbutviablevirus[36,37].AsshowninFigure 8A, Melissa officinalisgavesimilarinhibitioncurvesforwildtypeandHSV1-1(KOS)∆gC2-3with1µl/mlinhibitingplaqueformationby90% in both cases. This suggests that a component of extractfromMelissa officinalisdoesnotbindtogC,butrathermaybeinteractingwithgBonthevirion.

AgBdeficientHSV1isnotviable,sotoexaminetheroleofgB,his-taggedHSV1gBandgDweretransientlyexpressedinVerocellsandpurifiedbybinding to a 6Xhis-tag antibody-agarose resin.Melissa officinalisextractwasincubatedwithresinaloneorwithresincontainingboundgBorgD,andtheresultingsupernatant(unboundextractcomponents)wereremovedandtestedfortheability to inhibit HSV1plaque formation.Figure 8B shows thatbothresin-boundMelissa officinalisextractandextractincubatedwith gD-resin exhibited similar inhibition of plaque formation.However,extractincubatedwithgB-resinlostapproximately30-40%oftheHSV1inhibitoryactivity.TheseresultssuggestthattheHSV1 inhibitory componentpresent inMelissa officinalis couldbedepletedbybindingtogB.Cumulatively,theseresultssupport

Figure 5 Melissa officinalis inhibitsHSV1attachmenttocells.AplaquereductionassaywasperformedwithincreasingconcentrationsofMelissa officinalis orvehicleina(A)cellattachmentassay,(B)celluptakeassayasdescribed.InPart(C),cellsorHSV1werepreincubatedwithMelissa officinalis priortoinfectionfollowedbyaplaqueassay.InPart(D),HSV1virionswereincubatedwithheparin-agaroseinthepresenceofincreasingconcentrationsofMelissa officinalis followedbyWesternblotanalysis(forgB)oftheresinboundmaterial.

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thatMelissa officinalis inhibits HSV1 attachment to the cell bybindingtoandblockinggBreceptorinteractionwiththecell.

Higher concentrations of Melissa officinalis can inhibit cell to cell spread HSV1 gB plays an essential role not only for viral attachment,but forcell-to-cell spreadaswell [48].To test for theabilityofMelissa officinalis to inhibit viral spread, Vero cell monolayerswereinfectedwithHSV1atanMOI=0.001,andat6hpifollowingbinding and uptake, the cells were washed followed by theaddition of increasing concentrations of Melissa officinalis orhumangamma-globulin.Infectionswereallowedtoproceedfor

3daysfollowedbystainingofthemonolayers.Although1µl/mlMelissa officinalis didnotpreventspreadanddestructionofthemonolayer, a concentration of 3 µl/ml resulted in only limitedCPEof the cellmonolayer (Figure 9A).At concentrationsof10and30µl/ml,noviralspreadofCPEwasobserved(Figure 9A). These results suggest that Melissa officinalis can inhibit bothHSV1 attachment and cell-to-cell spread, although a higherconcentrationisrequiredfortheinhibitionofcell-to-cellspread.As a control, this assaywas done in comparison to treatmentwith human gamma-globulin. Human gamma-globulin bindsfreevirionsontheFcreceptorwhichisformedfromacomplexofglycoproteinEandglycoprotein[49].Adosedependenteffect

Figure 6 Melissa officinalis inhibitsGFP-labeledHSV1frombindingtocells.(A)Confocalmicroscopy.AbindingassaywasperformedwithGHSVatanMOI=50followedbystainingwithWGA-AlexaFluor®594andDAPI.PanelsA,B,C,andDarefullcolor,andpanelsE,F,G,andHaregray-scalepanelsofGFPonly.Treatmentconditionsarelistedabovethepanels.(B)Quantitativeanalysisofvirionbinding.ThreefieldsfromPart(A)wereanalyzedwith10cellspersampleandquantitatedforthenumberofboundvirions.*indicatesp<0.0001.

Figure 7 HighdoseMelissa officinalis disruptsvirionstructure.HSV1virionswereleftuntreated,treatedwith0.5%Triton,orweretreatedwithincreasingconcentrationsofMelissa officinalis orvehiclefor1hourat37°C.Virionswerepelletedthrougha20%sucrosepad,resuspendedin1XSDSsamplebuffer,andanalyzedbyWesternblotforenvelope(gD)andtegumentproteins(VP16).

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Figure 8 Acomponent(s)ofMelissa officinalis bindstogBtoinhibitHSV1.(A)Verocellswereinfectedwith100-200pfuHSV1orHSV1deletedforthegCgene(HSV1-1(KOS)∆gC2-3)andtreatedwithincreasingconcentrationsofMelissa officinalis orvehicle.(B)His-taggedHSV1glycoproteins,gBandgD,wereexpressedinVerocellsandpurifiedusingananti-His–agaroseresin.Melissa officinalis extractwasincubatedwithanti-His–agaroseresinaloneorwithresinboundwithgBorgD.Theresinwaspelletedandthesupernatantassayedforvirusinhibitionusingaplaquereductionassay.

Figure 9 Melissa officinalis inhibitscell-to-cellspread.(A)VerocellswereinfectedwithHSV1atanMOI=0.001followedbytheadditionof

increasingconcentrationsofMelissa officinalis orhumangammaglobulinat6hpi.After3daysmonolayerswerestainedwithcrystalviolettoCPE.(B)TheassayinPart(A)wasrepeated,butattheindicatedtimepoints,cellswereharvestedandtiteredtodetermineplaqueformingunits.

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Figure 10 Melissa officinalis inhibitsalphaherpesvirusesandmembersofothervirusfamilies.(A)Verocellmonolayerswereinfectedwith

100-200pfuHSV1,HSV2,andEHV1withincreasingconcentrationsofMelissa officinalis.After3daysofincubationplaqueswerephotographed.(B)Appropriatecellmonolayerswereinfectedwith100-200pfuofvirusinthepresenceofincreasingconcentrationsofMelissa officinalis.Afterincubation,plaqueswerestainedwithcrystalvioletforquantitation.Leftpanel-HSV1(•),HSV2(ϒ),andEHV().Rightpanel-HSV1(•),VSV(ϒ),EMCV(ρ),VACV(λ),ADENO(),REO(ο),andSV40(|).

ofhumangammaglobulinwasobservedwheredosesfrom0.1-3.0%producedagradientofreducedCPE.ThisisaverydifferentresultthanthatobservedwithMelissa officinaliswhereagradienteffectwasnotobservedwithfullCPEoccurredat1µl/ml,whileat 3 µl/ml the monolayer had only minimal CPE. To quantifythis effect,Figure 9B showstitersmeasured from infected cellmonolayers treated with 3 µl/mlMelissa officinalis or vehicle.Atall-timepointsmeasuredover thecourseof3days,Melissa officinalis inhibitedviral replicationby4-5 logsascompared tovehicle.

Melissa officinalis inhibits the replication of other virusesTheabilityofMelissa officinalistoinhibitotherrelatedmembersof the alpha herpes virus familywas tested by plaquingHSV1,HSV2,andequineherpesvirus1(EHV1)onVerocellmonolayerswithincreasingconcentrationsofextractfromMelissa officinalis. HSV2wasslightlymoresensitivetoMelissa officinalisascomparedtoHSV1(Figure 10A and B),whereasEHV1wasintermediateinsensitivity.InFigure 10A,theinhibitionofviralinducedCPEcouldeasilybeobserved inHSV1 infectedcellsat6µl/ml,HSV2at2µl/ml,andEHV1at18µl/ml.Toquantitatethis inhibitionmoreaccurately,theplaquereductionassay,showninFigure 10Btoppanel,demonstratestheID50forHSV1at0.8µl/ml,HSV2at0.2µl/ml,andEHV1at6.0µl/ml.

A number of additional virus families were tested for theirsensitivity to Melissa officinalis using various prototypeviruses. Vaccinia virus (VACV-Copenhagen, Orthopoxviridae)had similar sensitivity as HSV-1 to increasing concentrationsof Melissa officinalis, whereas vesicular stomatitis virus (VSV,Rhabdoviridae),adenovirus (ADENO,Adenoviridae),andsimian

virus 40 (SV40, Papovaviridae) had intermediate sensitivity toMelissa officinalis with ID50 ranging from 3.0-7.5 µl/ml (Figure 10B). Structurally, these intermediately sensitive viruses arebothenvelopedandnon-enveloped.Encephalomyocarditisvirus(EMCV, Picornaviridae) and reovirus (REO, Reoviridae) wereunaffectedbythepresenceofMelissa officinalisduringinfection(Figure 10B).

DiscussionThisstudysoughttofurtherdefinetheantiviralactivityassociatedwith Melissa officinalis. As shown, Melissa officinalis stronglyinhibitedHSV1-inducedCPEandplaque formation. Theextractwas relatively nontoxic to cells with a high SI of 327, therebysupportingitspotentialforuseasatherapeutic.Astanietal.andNolkemperetal.havereportedSIsof875and2200,respectively,forextractsofMelissa officinalis.Bothgroupsresuspendedtheirdriedmaterial in boilingwater rather than glycerinwhichwasused inourcurrentstudy [23,34].Thesepreviousresultsagreewithourresultssincecelltoxicitywithglycerinalonewasnearlyidenticalastheglycerin-extractedMelissa officinalis.

HSV1 virionswere shown to be stable up to 1 µl/ml followingincubationwithextractfromMelissa officinalis,howeverat3µl/ml, thevirionsbecomedisruptedasevidencedby lossofbothenvelopeandtegumentproteins.Thereforeitappearsthattwophenomena may be occurring with the extract from Melissa officinalis: inhibition of HSV1 binding at low concentrations (≤1µl/ml), anddisruptionof virion structureat≥3µl/ml. ThesetwophenomenaareevidencedintheconcentrationsofMelissa officinalis required to inhibit HSV1 in the binding and uptakeassays.InthebindingassayHSV1waspreincubatedwithMelissa officinalis followed by infection of cell monolayers with the

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mixture at 4°C.A concentrationof 0.3µl/mlMelissa officinalis resultedina60%reductioninplaqueformationandat1µl/ml,a 90% reduction. At these concentrations of extract, theHSV1virions were stable, so the effect ofMelissa officinalis was todirectlyinhibitbindingofvirionstothecell.TheabilityofMelissa officinalistoactdirectlyonvirionstopreventbindingtothecellhasalsobeensuggestedbyNolkemperetal.andAstanietal.[23,34].Alternately, intheuptakeassay,HSV1waspreboundtocellsat4°CfollowedbytheadditionofMelissa officinalis. Dosesbelow3µl/mldidnotaffectviralplaqueformation,butaconcentrationof3µl/mlwasabletoinhibit74%ofplaqueformation.Thisresultmaysuggestthatfollowingvirusbinding,ahigherconcentrationofMelissa officinalismayleadtodisruptionofthevirusparticlesalreadyboundtothecell.Therefore,Melissa officinalis inhibitsvirionbindingtothecellatlowerconcentrations,butinhibitionofuptakerequiresaconcentrationthatdisruptsvirionstructure.

The direct inhibition of HSV1 virions by Melissa officinalis is also supported by the data in the preincubation assay (Figure 5C).When increasingconcentrationsofMelissa officinaliswerepreincubated with cell monolayers followed by infection withHSV1,nosignificantreductioninplaqueformationwasobserved.However, when HSV1 virions were preincubated with Melissa officinalis followed by infection of cell monolayers, 0.3 µl/mlinhibitedplaque formationby42%.This supportsandsuggeststhatcomponentsinMelissa officinalisbinddirectlytothevirusandpreventattachmenttothecell.Inaddition,immunofluorescencedatashowedthat0.3µl/mlinhibitednearly80%ofvirionsfrombindingtothecells.

The initial stepofHSV1 inbinding toa celloccurswhenvirus-encodedglycoproteins,gBorgC,bindtoheparansulfatemoieties[36,37].Ithasbeenfoundthatsolubleheparin,whichisrelatedinstructure,canbeusedtoinhibitHSV1bindingtothecell[47,50].Inaddition,wefoundthatextractsfromMelissa officinaliscouldinhibitHSV1binding toheparin-agarose in vitro.This inhibitionoccurred at the lower concentrations atwhich virion structureremainedintact(0.3and1µl/ml).

As mentioned, the HSV1 gB and gC are involved in initial cellattachment. WtHSV1 and HSV1 deleted of gC were similarlyaffectedbyMelissa officinalisextractsuggestingthegCatleastisnotthesolecomponenttargetedtheactivebotanicalconstituent.More importantly, resin-boundgBwasabletopartiallydepletethe inhibitorycompound(s) fromtheMelissa officinalisextract.This keyexperiment supports ourprevious results and suggestthattheactivecompound(s)presentinMelissa officinalisbindsdirectly to gB to inhibit virus binding to cells. Density gradientanalysisofvirionsincubatedwith1µl/mlMelissa officinalis did notcauseanincreaseindensityduetocomponentsofMelissa officinalis binding to virions as seen previously with HIV (datanotshown)[51].This lackofshiftindensitymaybeduetotherelativelylowmolaramountsofgBpresentonHSV1virions[52].

Murine L-cell fibroblast cell lines that are deficient in heparansulfate (Gro2C) or are deficient in both heparan sulfate andchondroitinsulfate(sog9)havepreviouslybeenreported[53,54].When these cells were tested under single cycle conditions, aconcentrationof1µl/mlMelissa officinalisinhibitedHSV1yieldsby 90% in each cell line tested, regardless of the presence of

heparansulfateand/orchondroitinsulfate(datanotshown).ThisisinagreementwithdatafromBenderetal.thatshowsgBcanbindcellsindependentlyofheparansulfatebyusingalternativereceptors [54-58]. This indirectly supports that the inhibitorycomponent of Melissa officinalis likely binds to gB therebyprevents interactions with either heparan sulfate or otherstructurallyrelatedcellularreceptors.

WhencomparingotheralphaherpesvirusesfortheirsensitivitytoMelissa officinalis,HSV2appeared tobe themost sensitive,followed closely byHSV1, and EHV1 beingmore intermediate.All threevirusesutilizeheparinsulfate forcellattachment.ThereducedactivityagainstEHV1wassurprising,butmayberelatedtostructuraldifferencesintheviralgB.ForHSV1,Melissa officinalis also inhibitedcell-to-cell spreadof thevirus.Therequirementsfor virion binding/entry and cell-cell fusion are not identical,howevergD,gHgLandgBareessentialforbothprocesses[48].The inhibitoryactivityofMelissa officinalis targetinggBagreeswith itseffectonbothviral attachmentandcell-to-cell spread.HigherdosesofMelissa officinalis wererequiredtoinhibitcell-to-cellspread,butthismayberelatedtogBmechanismsofactionoraccessibilityduringviralattachmentversuscell-cellfusion.

Whenavarietyofotherviralfamiliesweretestedfortheabilityof Melissa officinalis extract to inhibit replication, strong inhibitoryactivitytowardsVACVwasobserved.VACVinfectioncanbeinhibitedbysolubleheparansulfate,inagreementwiththeabilityofMelissa officinalis to inhibit infection[59].VSV,adenovirus and SV40 were moderately inhibited by Melissa officinalis.VSVhasnotbeenshowntobindtoheparansulfate,however it requires a charged interaction for binding to thecell, and negatively chargedmolecules have been shown toinhibitinfectionpossiblyexplainingthispartialinhibition[60].Adenovirushasbeenshowntobindheparansulfateaswellasvariousothercellsurfacemolecules[61,62].Thisbroadrangeof cell surface receptors may support the partial inhibitionobserved. For SV40, viral binding involves the cellularglycolipid,GM1,whichcontainsasinglesialicacidmoiety[63].It is possible again that Melissa officinalis partially inhibitsinteraction of the virus particlewith this receptor. The finaltwo viruses in our study thatwere not inhibited byMelissa officinalis,EMCVandReovirus,bothbindtosialicacidonthesurface of the cell [64-66]. The results suggest thatMelissa officinalis was not able to bind to these viruses and inhibitcell attachment. Together, the viral family results supportthatcomponent(s)ofMelissa officinalisareabletobindviralenvelope or capsid proteins and prevent interaction withcellularheparansulfateorrelatedreceptors.

AcknowledgementsThe authors would like to thank Gary H. Cohen and RoselynJ Eisenberg for their generous gift of L, Gro2C, and sog9, Dr.VladimirChouljenko for theplasmids,pPEP98 (gB)andpPEP99(gD),DavidBloomforHSV1KOSandforantibodiestoICP4,andtheVeteran’sAdministrationHospitalResearchCenter(Phoenix,Arizona)foruseoftheirconfocalmicroscopyfacility.

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