Research Article Sialic Acid Expression in the Mosquito

Research ArticleSialic Acid Expression in the Mosquito Aedes aegypti andIts Possible Role in Dengue Virus-Vector Interactions

Jorge Cime-Castillo1 Philippe Delannoy2 Guillermo Mendoza-Hernaacutendez3

Veroacutenica Monroy-Martiacutenez1 Anne Harduin-Lepers2 Humberto Lanz-Mendoza4

Fidel de la Cruz Hernaacutendez-Hernaacutendez5 Edgar Zenteno3

Carlos Cabello-Gutieacuterrez6 and Blanca H Ruiz-Ordaz1

1Molecular Biology and Biotechnology Department Biomedical Research Institute National University of Mexico (UNAM)04510 Mexico City Mexico2Structural and Functional Glycobiology Unit UMR 8576 CNRS University of Sciences and Technologies of Lille59655 Villeneuve drsquoAscq France3Biochemistry Department Faculty of Medicine UNAM 04510 Mexico City Mexico4CISEI National Institute of Public Health 62100 Cuernavaca MOR Mexico5Infectomics and Molecular Pathogenesis Department CINVESTAV-IPN 07360 Mexico City Mexico6Virology Department National Respiratory Institute (INER) 14050 Mexico City Mexico

Correspondence should be addressed to Blanca H Ruiz-Ordaz bhrounammx

Received 25 July 2014 Accepted 24 September 2014

Academic Editor Michael J Conway

Copyright copy 2015 Jorge Cime-Castillo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans DF is caused by the four denguevirus (DENV) serotypes which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infectionreplication and viral transmission (vector competence) Mosquito saliva also plays an important role during DENV transmissionIn this study we detected the presence of sialic acid (Sia) inAedes aegypti tissues which may have an important role during DENV-vector competence We also identified genome sequences encoding enzymes involved in Sia pathwaysThe cDNA forAedes aegyptiCMP-Sia synthase (CSAS) was amplified cloned and functionally evaluated via the complementation of LEC29Lec32 CSAS-deficient CHO cells AedesCSAS-transfected LEC29Lec32 cells were able to express Sia moieties on the cell surface Sequencesrelated to 120572-26-sialyltransferase were detected in the Aedes aegypti genome Likewise we identified Sia-120572-26-DENV interactionsin different mosquito tissues In addition we evaluated the possible role of sialylated molecules in a salivary gland extract duringDENV internalization in mammalian cells The knowledge of early DENV-host interactions could facilitate a better understandingof viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission

1 Introduction

Dengue fever (DF) is themost important and rapidly expand-ing arthropod-borne viral disease in tropical areas Denguevirus (DENV) infection affects more than 100 million peopleworldwide each year and 25 billion people live in areasof risk [1] DF is caused by any of the four antigenicallydistinct dengue virus serotypes (DENV 1ndash4) which aretransmitted to humans by the hematophagous mosquitoesAedes (Ae) aegypti and Ae albopictus The recent increase in

DF and dengue hemorrhagic feverdengue shock syndromenow known as severe dengue is associated with the vectorrsquosexpansion to new geographic areas [2] Severe dengue is ahighly pathogenic disease so the development of a denguevaccine is a high priority for protecting people at risk butno safe vaccine is available at present Therefore mosquitocontrol is the primary option for preventing dengue out-breaks [3] Ae aegypti females have a key role in DENV-vector competence which refers to the vectorrsquos permissive-ness to infection replication and viral transmission [3 4]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 504187 16 pageshttpdxdoiorg1011552015504187

2 BioMed Research International

The female mosquito acquires DENV from an infectedperson during blood feeding The virus undergoes its firstreplication cycle in the mosquito midgut before spreadinginto the hemocoel and finally infecting the salivary glands(SGs) The transfer of infectious saliva into a human host(during a new blood feeding) is a key event during the DENVtransmission cycle [4 5]Thus it is very important to identifythemolecules involved in the DENV-SG relationship becausemosquito saliva is rich in glycoproteins that participate indifferent host responses (platelet activation swelling itchingand inflammation) as well as the binding and transportof vector-borne pathogens to host tissues thereby allowingpathogens to infect and evade the host immune response[5] In an ample range of disease models including vari-ous hosts mosquito species and arthropod-borne virusesmosquito saliva andor mosquito feeding are associated witha potentiation of the arbovirus (arthropod-borne) infectionHost infection via vector saliva leads to an increase in viraltransmission host susceptibility disease progression andmortality [6] The potential for mosquitoes to influence thecourse of West Nile virus (WNV) disease was investigatedby assessing pathogenesis in the presence or absence ofmosquito saliva [6] Likewise in vitro and in vivo modelsof saliva-mediated enhancement of DENV infectivity havebeen reported [7] but it is uncertain whether Aedes salivaglycosylatedmolecules contributes to DENV tissue infectionThe Aedes sialome includes 136 putative secretory proteinswhich couldmodify host responses [8]DuringDENV-vectorinfection the main genes upregulated in Ae aegypti arerelated to carbohydrate expression [9] but the roles of glycansin vector competence are currently unknown In additionit is known that certain glycosidases affect the binding ofDENV to mammalian (green monkey kidney and Vero) andmosquito (C636 and AP61) cell surfaces [10] Previously itwas reported that 120573-glucosidase sialidase and heparinasereduce DENV attachment to mammalian cells but not toinsect cells [10] and the inability of sialidase to affect DENVbinding to insect cells is associated with a lack of mosquitosialyltransferase (ST) which is capable of transferring sialicacid (Sia) residues to mosquito glycoproteins [11] Moreoverthe occurrence of Sia in mosquito tissues is also unknownHowever the genetic and biochemical capacity for sialylationin Drosophila melanogaster supports a hypothesis that insectsialylation is a specialized and developmentally regulatedprocess in insects [12ndash16] This process is involved in theregulation of neural transmission in the nervous system ofD melanogaster [17 18] It is well known that sialylatedglycoproteinsmodulatemany important biological processesincluding cellular andmolecular recognition subcellular andcellular trafficking intercellular adhesion and signaling andmicrobial attachment among others [19] In the presentstudy we detected the presence of a functional cytidinemonophosphate- (CMP-)Sia synthase (CSAS) in Ae aegyptiand we also demonstrated that DENV recognizes 120572-26-linked Sia structures on the surface ofmosquito tissues whichmay play key roles during early DENV-vector interactionsFurthermore we found that DENV is capable of interactingwith secretory Sia-glycoproteins which may be involved in

successful DENV-host tissue transmission To our knowl-edge these are the first demonstrations of the functionalexpression of anAedesCSAS and the presence of Sia moietiesin mosquito tissues which may have important biologicalconsequences for DENV-vector competence Knowledge ofspecific early DENV-mosquito interactions could facilitatea better understanding of viral tropism and pathogenesisto allow the development of new effective strategies for thecontrol of DENV transmission as well as the improvementof antiviral agents and vaccines

2 Materials and Methods

21 DENV Propagation and Titration DENV New GuineaC strain serotype 2 (DENV-2 kindly donated by Dr DuaneGubler CDC Fort Collins CO USA) was propagated inC636 cells which were grown at 28∘C in supplementedminimal essential medium (MEM) Confluent monolayerswere infected for 2 h at a multiplicity of infection (MOI)of 1 and incubated for 5ndash7 days at 28∘C in a 5 CO

2

atmosphere until cytopathic effects were observed beforetitrating in a lytic plaque assay using LLC-MK2 cells asdescribed previously [20] The virus titer was expressed asplaque-forming units (pfu) per milliliter

22 Ae aegypti Maintenance Salivary Glands Midgut Iso-lation and Tissue Extracts Female Ae aegypti mosquitoeswere cultured in an insectarium at the Center for Infec-tious Disease Research (CISEI-INSP) Mexico The SGs andmidguts of female mosquitoes (at least three days old and fedonly with water) were dissected using a microneedle placedin sterile tubes in groups of 20 pairs with 20120583L of phosphate-buffered saline (PBS) and kept at minus75∘C The tissues werelysed during five freeze-thaw cycles using liquid nitrogenand sonicated (ultrasonic 8849-00 Cole-Parmer IL USA)for 10min before centrifugation at 3500 rpm to obtain tissueextracts The protein concentration was determined usinga micro-BCA (bicinchoninic acid) assay (Pierce USA) at562 nm with a spectrophotometer (Multiskan Ascent 354Thermo Labsystem UK)

23 Ae aegypti Saliva Collection Ae aegypti saliva wascollected as described by Almeras et al [21] with a smallnumber of modifications Female mosquitoes were sedatedfor 1min at 4∘C and the proboscis of each mosquito wasplaced in a plastic pipette tip containing mineral oil After1 h salivation at room temperature (RT) the liquid wascollected from the tip and the saliva from 20 mosquitoeswas pooled before centrifugation at 10000 rpm The proteinconcentration was estimated using a micro-BCA assay

24 Carbohydrate Determination in Ae aegypti SalivaryGlands The salivary glands of femaleAe aegyptimosquitoeswere dissected as described above and the SG monosac-charides were analyzed according to Kamerling et al[22] by GCMS as trimethylsilyl methyl glycosides (bythe Structural and Functional Glycobiology Unit of theUniversity of Sciences and Technologies of Lille France)Briefly dry samples were methanolized in methanolHCl

BioMed Research International 3

05N N-reacetylated and trimethylsilylated in a mixtureof NO-Bis(trimethylsilyl)trifluoroacetamide and pyridine(1 1) before injection into a gas chromatographwith a BPX7012m times 022mm diameter column (Chrompack)

25 Identification of Sia in Ae aegypti Midguts by High-Performance Liquid Chromatography (HPLC) Midguts werehomogenized in water lyophilized and incubated in 1mL01M TFA at 80∘C for 2 h The samples were centrifugedat 5000 rpm for 15min and two volumes of cold ethanolwere added to the supernatant To obtain exact analyticaldata and to avoid false-positive results the lyophilized Siaswere dried resuspended in 100120583L of water and passedsuccessively through 50 times 2 (200 times 400 mesh) and 50 times 8(25times50mesh)Dowex (100 120583L) anion exchange columns (Bio-RadMarnes-la-Coquette France)This sequential cation andanion exchange chromatography process was described indetail in a previous study [23] The columns were elutedwith three volumes of water The total volume was drieddiluted in one volume of 001M trifluoroacetic acid (TFA)and analyzed by HPLC using a Hewlett-Packard model 1100liquid chromatography system (Palo Alto USA) as followsIn the HPLC analysis Sia was derivatized using 12-diamino-45-methylenedioxybenzene according to Hara et al [24]and separated isocratically in a C-18 reverse phase Sep-PaKHPLC column (250 times 46mm 5 120583m Vydac Hesperia CAUSA) using a solvent mixture of acetonitrilemethanolwater(7 9 84) followed by identification based on the elutionpositions of standard Neu5Ac derivatives

26 Lectin Histochemistry of Ae aegypti SGs andMidguts Aeaegypti SGs and midguts were placed on slides and fixedand the tissues were then blocked with 2 bovine serumalbumin (BSA) for 30min at RT washed with PBS for 5minand immersed in PBS-Triton X-100 (02) for 10min Nextthey were washed with PBS-Ca2+ (1mM) for 10min andincubated with different biotin-conjugated lectins that isMaackia amurensis lectin (MAA) Sambucus nigra agglutinin(SNA) or Lens culinaris hemagglutinin (LCH) (EY Labo-ratories Inc USA) at 1 100 dilutions for 2 h at 37∘C Theslides were washed with PBS for 10min and incubated inthe dark with ExtrAvidin-fluorescein isothiocyanate (FITCZymed Inc USA) at 1 60 The tissues were then rinsed withPBS-Ca2+ (1mM) for 5min and with deionized water for5min Finally the samples were mounted with Vectashield410158406-diamidino-2-phenylindole (DAPI Vectashield VectorLaboratories CA USA) and visualized using a Leica DMfluorescence microscope (DCF-300FX digital camera LeicaMicrosystems Digital Imaging Germany) To evaluate SNA-specific binding mosquito SGs and differentD melanogastertissues fixed on slides were pretreated with 05 IU Clostrid-ium perfringens sialidase (Roche Diagnostics Germany) for30min at RT This sialidase was preincubated with caseinand resorufin-labeled according to Twining [25] to pre-vent protease activity Samples were incubated in the darkwith biotinylated SNA lectin (1 100) and streptavidin-FITC(1 60) The fluorochromes were analyzed in two channelsgreen for lectins and blue for nucleiThe gut SGs andmidgut

from D melanogaster were dissected fixed (as describedpreviously [26]) and incubated with SNA lectin or sialidaseFinally the images were digitized with the Leica IM1000 ver-sion 120 program (Imagic Bildverarbeitung AG GlattbruggSwitzerland)

27 DENV-Lectin Binding Assays SGs were fixed on slidesand incubated overnight with DENV (107 pfu) at 4∘C Thesamples were washed three times each for 10min usingPBS and incubated for 2 h at 37∘C with the anti-DENVprotein-E antibody (dengue type-2 virus MAB8702 Chemi-con International CA USA) at a dilution of 1 300 Nextthe samples were washed with PBS for 10min and incubatedfor 20min at RT in the dark with rhodamine-coupled anti-IgG antibody (Zymed Laboratories Inc USA) at a dilutionof 1 3000 In the competition assays SGs were incubatedwith lectins before the addition of DENV To evaluate thepossible participation of Sia in DENV-SG interactions aDENV-SG competition assay was performed where DENVwas preincubated for 1 h with soluble 200mM Sia (N-acetylneuraminic acid Sigma-Aldrich) or 1mM fetuin (DIGGlycan Kit Roche) before adding it to the SG Images wereacquired in three channels green for lectins red for anti-DENV and blue for nuclei

28 Trypsin and Sialidase Assays of SGs and GlycoproteinIdentificationUsing a Lectin Blot Assay SGswere treatedwith05 IU of C perfringens sialidase (Roche Applied ScienceUSA) for 30min or with 0075 trypsin (Sigma-AldrichInc USA) for 5 15 or 30min before the glands were fixedand incubated with DENV The SGs were incubated withSNA MAA or LCH lectins Finally images were obtainedas described earlier

29 SG Glycoprotein Detection by Blot Assay Glycoproteinsin the SG protein extracts were identified by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) witha polyacrylamide gradient of 4ndash20 which was then stainedto detect all carbohydrates using a Pro-Q Emerald 300Glycoprotein Gel Stain kit (Molecular Probes InvitrogenP21855) according to the supplierrsquos protocol The gel imagewas captured under a UV transilluminator (Kodak Gel Logic1550) For the lectin blot assay proteins were transferredto nitrocellulose membranes (Trans-Blot 162-0112 Bio-Rad)blocked with 1 BSA + 02 Tween-20 in PBS and washedThe membranes were incubated with biotinylated SNA orCanavalia ensiformis agglutinin (ConA EY LaboratoriesInc USA) at a dilution of 1 10 for 3 h at RT followedby streptavidin-horseradish peroxidase conjugate (43-4323Zymed Laboratories Inc USA) at a dilution of 1 3000 for1 h at RT The membranes were then washed with PBS andvisualized with luminol (Western Blotting Reagent sc-2048Santa Cruz Biotechnology USA) Finally the membraneswere exposed to a film (Kodak)

210 VirusOverlay Protein BindingAssays (VOPBA) VOPBAwas performed as described by Salas-Benito and del Angel[27] Briefly SG protein extracts or salivary proteins were


transferred to nitrocellulose membranes blocked (1 BSA +02 Tween-20 in PBS) for 1 h at RT washed three times withPBS and incubated overnight (4∘C) with DENV (107 pfu) in1 BSA in PBS + 1mM CaCl

2 The membranes were washed

with PBS and incubated for 35 h at RT with a monoclonalantibody against DENV protein E (MAB 8702 ChemiconInternational CA USA) at a dilution of 1 300 Next themembranes were washed twice with PBS + 50mMNaCl andincubated for 1 h at RT with a secondary anti-mouse IgGantibody (1 5000) coupled with peroxidase (81-6520 ZymedLaboratories Inc) Finally the membranes were washedtreated with luminol and exposed to film To evaluate therole of Sia residues in interactions with DENV the SGprotein extracts and saliva were pretreated with 05 IU of Cperfringens sialidase (Roche) for 1 h before the overlay assayas described earlier

211 DENV Infection ofMammalian Cells in the Presence of Aeaegypti SG Protein Extract The internalization of DENV inmammalian cells (LLC-MK2 and wild-type Chinese hamsterovary cells CHO) was assessed in the presence or absence ofSG extract protein where DENV was metabolically labeledwith [35S]-methionine at 37∘C for 1 h Confluent monolayersof mammalian cells were infected with labeled DENV at anMOI of 1 in the presence or absence of SG proteins extractedfrom 80 SGs which were pretreated (or untreated) with05 IU of C perfringens sialidase for 1 h at RT After infectionthe medium was removed and the cells were washed twicewith citrate buffer (10mM citric acid 005 Tween-20 pH60) and PBS to remove any nonspecifically associated virusafter the incubation period thereby avoiding counting virusthat was not internalized Cells were subsequently lysed andfixed on mats filters (Skatron Instruments UK) The [35S]-methionine level was measured using an LS6500 ScintillationCounter (Beckman Coulter USA)

212 LCESI-MSMS Analysis VOPBA protein bands wereselected for protein identification by mass spectrometry(MS) analysis The bands were carefully excised fromCoomassie Brilliant Blue-stained gel and prepared for liq-uid chromatography-electrospray ionization tandem massspectrometry (LC-MSMS) Briefly individual protein bandswere destained reduced carbamidomethylated digestedwith trypsin and extracted from the gel using a standardin-gel digestion procedure [28] The volumes of the extractswere reduced by evaporation in a vacuum centrifuge atRT before adjusting to 20120583L with 1 formic acid PeptideMS analysis was performed using a 3200 QTRAP System(Applied BiosystemsMDS USA) which was equipped witha nanoelectrospray source and a nanoflow LC system (1100Nanoflow Pump Agilent Waldbronn Germany) Mass tun-ing of the hybrid triple quadrupole linear IT spectrometerwas performed using [Glu1]-fibrinopeptide B Sample digestswere injected into a Zorbax 300SB C18 column equilibratedwith 2 ACN and 01 formic acid and separated usinga linear gradient of 2 to 7 CAN with 01 formic acidover an 80min period at a flow rate of 300 nL minminus1 Theinterface heater used for desolvation was held at 150∘C

and the spray voltage was 24 kV Spectra were acquired inthe automated mode by information-dependent acquisitionPrecursor ions were selected in Q1 using the enhancedMS mode The scan ranges for EMS were set to 400ndash1500 and 4000 amu sminus1 Selected ions were subjected to anenhanced resolution scan at a low speed of 250 amu sminus1 overa narrow (30 amu) mass range followed by an enhancedproduct ion scan (MSMS) The precursor ions were frag-mented by collision-activated dissociation in the Q2 col-lision cell using rolling collision energy The fragmentedions were captured and mass analyzed in the Q3 linearIT Database searches (Swiss-Prot NCBInr or MSDB) andprotein identification were performed using the MASCOTprogram (httpwwwmatrixsciencecom) with trypsin plusone missed cleavage and carboxyamidemethylation as a fixedmodification and methionine oxidation as a variable modi-fication using a mass tolerance of 05Da for the precursorMWs and 03Da for the fragment MWs The criteria used toaccept a protein hit as a valid identification were two or moretryptic peptidematches with the protein sequence and at leastone peptide with 119875 lt 005

213 Analysis of the Protein Glycosylation Sites The sequenceobtained from the MASCOT database was analyzed withGlycomod [29] which is available at httpwwwexpasychtoolsglycomod This program explores the mass values ofions obtained experimentally with MALDI-ToF and theirrelationships with sequences in the MASCOT databaseThe search parameters specified N-glycosylated and O-glycosylated proteins with modifications of oxidized methy-lation and cysteine-treated iodoacetamide using a masstolerance of 01 Da

214 Ae aegypti RNA Purification Groups of 25 femalemosquitoes were homogenized and sonicated with RNAse-free water The lysates were passed through a 09mm needleRNA extraction was performed using a Nucleospin RNA IIkit (Macherey-Nagel Germany) and the RNA quality wasevaluated using Agilent RNA Nano 6000 chips (Agilent 2100Bioanalyzer)

215 Ae aegypti CSAS and ST Gene Synthesis BLink andBLAST searches for CSAS and ST genes were performedusing the NCBI tBLAST algorithm based on the CSAS(gi|24667125) and ST (gi|24762715) sequences of Dmelanogaster Putative CSAS (XP 001663017) and ST (XP001649590) genes were identified in the Ae aegypti genomeand confirmed by VectorBase (httpswwwvectorbaseorg)as AAEL012868 and AAEL014772 respectively cDNAsynthesis was performed using 200 ng of RNA template(QPCR cDNA kit Stratagene USA) with random primersFive microliters of cDNA was used in a 25 120583L PCR reactionwhich was amplified with Taq DNA polymerase (ThermoFisher Scientific) as follows 95∘C for 5min 38 cycles at94∘C for 1min 50∘C for 1min 72∘C for 15min and 72∘C for10min holding at 4∘C The following primers were used forAeCSAS gene synthesis 51015840aedsy (51015840GTT GAA TTC CATGCG GCT AGT TTT GAT 31015840) 31015840aedsy (51015840AAT GGT ACC


TTA TTC TAC TGT GGA TCC 31015840) 51015840aedtr (51015840CAC AAGCTT ATG TTG CGT GAC CTT TCG 31015840) 31015840aedetr (51015840CTAGGT ACC TCA ACA TCC ACT GTT GCT 31015840) 51015840Act(51015840TGG TTA CTC GTT CAC CA 31015840) and 31015840Act (51015840GGCATA CAG ATC CTT TCG GA 31015840)

The forward primer 51015840aedsy included an EcoRI site andthe first six codons ofAeCSASThe 31015840aedsy primer containeda KpnI site and the last six codons of AeCSAS The 51015840aedtrforward primer contained a HindIII site and the first sixcodons of the hypothetical Ae aegypti ST sequence and31015840aedtr included a KpnI site and the last six codons of thesame sequence The Ae aegypti actin gene was used as ahousekeeping control

216 Ae aegypti CSAS cDNA Cloning and Sequencing TheCSAS PCR product was cloned using a Topo vector (Invit-rogen) and transformed into Escherichia coli strain DH5120572The cloned cDNA was evaluated by PCR using M13 forward(ndash20) and reverse primers The CSAS cDNA was nicked atthe EcoRI and KpnI sites and subcloned using a p3XFlag-CMV-10 (Sigma-Aldrich) vector The plasmid sequence wasconfirmed by PCR using the primers 51015840p3 FLAG (51015840-GTTGACGCAAATGGGCGGTAG-31015840) and 31015840p3 FLAG (51015840-CTTGCCCCTTGCTCCATACCAC-31015840) as follows 96∘C for5min 38 cycles at 96∘C for 45 s 50∘C for 45 s 72∘C for 1minand 72∘C for 10min holding at 4∘CThe 786 bpCSASproductwas sequenced (Genoscreen Lille France)

217 Complementation of CSAS-Deficient Cells with AeCSASWild-typeCHOcells and LEC29Lec32 cells whichwere defi-cient in CMP-Neu5Ac synthase were grown in MEM con-taining 10FBS in 5CO

2at 37∘COnemillion LEC29Lec32

cells were transfectedwith lipofectamine reagent (Invitrogen)using 5120583g of the p3XFlag-CMV-10 vector with the AeCSASinsert or the empty vector as a control Cells were harvestedat 36 h posttransfection Ae aegypti Sia expression was eval-uated by FACS analysis Cells were detached and incubatedfor 1 h at 4∘C with biotin-conjugated MAA washed andincubated for 1 h on ice with Alexa Fluor 488 conjugatedstreptavidin (Invitrogen) Appropriate isotype and secondaryantibody controls were used In the FACS analysis 10000cells were analyzed using a FACSCalibur system (BectonDickinson USA) AeCSAS expression was also evaluated byhistochemistry that isWTCHOand LEC29Lec32 cells weregrown on slides and transfected as described previously Cellswere incubated with MAA lectin and Alexa Fluor conjugatedantibody and stained in parallel with DAPI

218 Hemagglutination Assay with DENV The assay wasperformed as described by Goldsmith (see [30] and Casalsand Brown [31]) DENV was propagated in C636 cellspurified by ultracentrifugation (see Methods in the paper)and suspended in borate solution (pH 9) Borate solutionwas used as the negative control In a microtiter plate aseries of twofold dilutions of the viral stock was generatedwhichwas followed by the addition of a suspension of chickenerythrocytes (4 in borate solution) and incubation of thesamples for 1 h at 4∘C The hemagglutination activity was

expressed as a titer defined as the reciprocal of the maximaldilution that gave positive hemagglutination A parallel assaywas performed using the influenza virus

219 Sialidase-Treated Erythrocytes Sialidase-treated ery-throcytes were obtained according to Sano and Ogawa [32]Briefly native chicken erythrocytes (10 vv) were mixedwith an equal volume of the incubation buffer (01M acetatebuffer containing 1mM CaCl

2 pH 55) containing sialidase

from Clostridium perfringens (1 UmL) which was preincu-bated with casein and resorufin to prevent protease activityThe sample was incubated at 37∘C for 1 h with occasionalcareful shaking The cells were washed by centrifugationusing cold PBS (pH 7) and stored as a 10 suspension at4∘C until use The HA assay was carried out as previouslydescribed A parallel assay was performed using the influenzaA virus

220 Statistical Analysis Data were expressed as the meanand standard deviation and compared using aMann-Whitney119880 test with Statistical Analysis Software version 8 (SASInstitute USA) The significance level was set at 119875 lt 005To identify the D7 protein in MASCOT and the score foran MSMS match was based on the absolute probability(119875) that the observed match between the experimental dataand the database sequence was a random event We used aprobability-based MOWSE score that is the reported scorewasminus10 log (119875) where119875was the probability that the observedmatch was a random event and the protein scores weresignificant at 119875 lt 005

3 Results

31 Identification of Sia in Ae aegypti Mosquito Tissues andGenes Involved in the Sia Synthesis Pathway The total carbo-hydrate composition of theAe aegypti SG protein extract wasdetermined by gas chromatography which showed that themost abundant monosaccharide was N-acetylgalactosaminewith an average of 170120583g per 10 salivary glands followedby mannose (84 120583g) N-acetylglucosamine (42120583g) galactose(16 120583g) and Sia (Neu5Ac with 7120583g) We also assessed thepresence of Sia in midguts using HPLC by referring tothe retention times of standard Sia derivatives [33] Siawas determined at a concentration of 14120583g per singlemidgut As a consequence of the presence of Sia in differentmosquito tissues we evaluated the possible existence ofgenes encoding enzymes involved in Sia synthesis pathwaysThe sialylation process requires the biosynthesis of glycosyl-nucleotide cytidine 51015840-monophosphate-N-acetylneuraminicacid (CMP-Neu5Ac) by CSAS and enzymes from the STfamily which transfer Sia to a glycoprotein or glycolipidacceptor substrate Therefore using the available genomedatabase of D melanogaster we searched for the amino acid(aa) sequences of both enzymes that is CSAS (gi|24667125)and DSialT6 ST (gi|24762715) and we performed BLASTand BLink analyses of the Ae aegypti genome using theNCBI genome database We detected hypothetical sequencesfor both proteins that is CSAS (XP 001663017 AeCSAS)and ST (XP 001649590 AeST) in the Ae aegypti genome


which were validated in the VectorBase database The Aeaegypti ST gene sequence was identified and associated withthe ST6Gal 12057226-sialyltransferase (ST6Gal) family which isclosely related to D melanogaster DST6 and orthologous tothe common ancestral gene thatwas present before the split ofST6Gal I and ST6Gal II [34]We used these sequences to gen-erate a complementary DNA (cDNA) that comprised 786 bpfor AeCSAS and another of 1396 bp for AeST (Figure 1(a))Likewise we obtained Ae aegypti cDNAs for AeCSAS andAeST from the SGs and midguts (Figure 1(b)) The AeCSAScDNAwas cloned into the p3XFlag-CMV vector Two clonesthat is C4 synthase and C8 synthase were sequencedanalyzed and compared with previously reported CSASsequences (See Figure S1 in SupplementaryMaterial availableonline at httpdxdoiorg1011552015504187) Both clonescontained the start point of an open reading frame for aprotein containing 261 aas with amolecular mass of 298 kDaand a theoretical isoelectric point of 672 We detected apolymorphism site in the AeCSAS gene (Figure 1(c)) Inclone 4 a point mutation from A (residue 183) to T changedan aspartic acid (D) residue into glutamic acid (E)

32 Evaluation of AeCSAS Complementation of CHO Sia-Deficient Cells To determine the functional activity ofAeCSAS a p3XFlag-CMV vector containing the AeCSASinsert was transfected into CHO LEC29Lec32 cells [35]which were deficient in CSAS expression and did not expresssialoglycoconjugates Sia expression was evaluated by a flowcytometry (FACS) assay using MAA which recognizes Sia in120572-23-linkages because CHO cells mainly express 120572-23-STs[36] We observed that AeCSAS-transfected cells expressed120572-23-linked Sia (Figure 1(d) blue line) at a similar level tothe parental CHO cells which were used as a positive control(Figure 1(d) magenta line) The intensity of fluorescence inthe nontransfected CHO LEC29Lec32 subpopulation wassimilar to that in the negative control (Figure 1(d) green andblack lines) In addition nearly 30 of the LEC29Lec32-transfected cells were able to express Sia (Figure 1(d) showsthe fluorescence intensity percentages) To confirm the func-tional activity of AeCSAS we tested for the presence of Siain AeCSAS-transfected CHO LEC29Lec32 cells using anaffinocytochemical assay with MAA lectin Sia expressionwas observed on the cell surface of AeCSAS-transfectedCHO LEC29Lec32 cells (Figure 1(e)) as shown by the FACSassay These results demonstrate the functional expression ofAeCSAS in Ae aegypti

33 DENV-Sia Interaction in Ae aegypti Tissues The Aeaegypti ST gene is related to the ST6Gal family [37] thuswe evaluated gene expression based on the presence of 120572-26-Neu5Ac moieties on the surface of mosquito tissues (SGhead and midguts) using affinocytochemistry and confocalmicroscopy assays with the lectin SNA which recognizesSia in 120572-26-linkages We observed strong SNA staining inthe differentmosquito samples (Figure 2(a))Dmelanogastertissues were used as the positive control and are well known[15] to express 120572-26-linked Neu5Ac moieties (Figure 2(b))No MAA binding was observed in Ae aegypti tissues whichindicates that Ae aegypti does not express 120572-23-ST (similar

to D melanogaster Figure S2) To validate the SNA bindingassay SGs were pretreated with C perfringens sialidaseand incubated with SNA lectin In the absence of sialidasetreatment strong SNA staining was observed in Ae aegyptimosquito andDmelanogaster tissues (Figures 2(a) and 2(b))However the SNAbinding decreased after sialidase treatmentof the mosquito and D melanogaster tissues (Figure 2(c))

SG is the main tissue where DENV is replicated andamplified in the mosquito before transmission to its ver-tebrate host thus we evaluated the possible role of Sia inDENV-SG interaction We performed a binding assay withAe aegypti SG in the presence of different lectins (SNA LCHor ConA) Figure 3(a) shows that there was a positive DENV-SG interaction in the absence of SNA lectin However DENVbinding decreased when 120572-26-Sia residues were blockedwith SNA (Figure 3(b)) whereas the blocking of mannoseresidues with ConA or LCH did not modify the DENV-SGinteraction (Figure 3(b) DENV-midgut interaction FigureS3) To confirm the possible role of Sia during DENV-SGbinding SGs were pretreated with C perfringens sialidase at30min prior toDENVadditionWe observed a large decreasein the DENV-SG interaction when the SGs were pretreatedwith sialidase (Figure 3(c)) To evaluate the specific roleof Sia in DENV-SG binding we performed a DENV-SGcompetition assay using free Neu5Ac and sialylated glyco-protein fetuin We observed that the DENV-SG interactiondecreased in the presence of fetuin and it was lost in thepresence of free Neu5Ac (Figure 3(c)) thereby suggestingthe involvement of Sia in DENV-SG recognition SGs werepretreated with trypsin for 5 15 or 30min to determinewhether the sialylated molecules related to DENV-SG wereproteins (Figure 3(d))The interaction with DENV decreasedafter 15min of incubation and it was abolished completelyat 30min These data suggest the possible participation ofsialylated glycoproteins in DENV tissue attachment

34 Detection of Ae aegypti SG Glycoproteins by Blot AssaysTo confirm the presence of total sugars in the SG proteinextracts from Ae aegypti and to characterize the putativeglycoprotein(s) that may recognize DENV we separated theSG proteins by electrophoresis and stained them to detectany carbohydrates The SG protein extracts were transferredto nitrocellulose membranes and subjected to a western blotassay The membrane was also incubated with ConA or SNAlectins (Figures 4(b) and 5(a) lane 9) For the control assaywe used a carbohydrate staining kit (Pro-Q Emerald 300Glycoprotein Gel Stain Kit Molecular Probes Figure 4(a)lane 1) andwe observed a range of glycoproteins from 29 kDato 116 kDa with more intense bands of 29 45 and 66 kDaWhen we incubated the SG protein extracts proteins withConA we observed a glycoprotein of 50ndash60 kDa which hasnot been identified previously with the carbohydrate stainingkit We also observed an increase in the intensity of the bandat 97 kDa Therefore these proteins could have containedmannose and glucose residues (Figure 4(b)) The interactionwith SNA produced several bands that ranged from 10 to97 kDa (Figure 5(a) lane 9) so these proteins could possessSia motifs In agreement we observed no significant changes


1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

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125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260

TCACTGAGAGTGGATTGCGTATTCAGCGTTACGAGAAGTTTTAAGCTAAGGTGGCGGAAG-S--L--R--V--D--C--V--F--S--V--T--R--S--F--K--L--R--W--R--K-

ATGCGGCTAGTTTTGATTCTGACGCTTCTCAGTGCACATCCAGCATTTGGTTTCCTTCAA-X--R--L--V--L--I--L--T--L--L--S--A--M--P--A--F--G--F--L--Q-

GAGAAAAGTGTGACGTGTCCAACATCCCCTGAAAGCATATCCAATGACAGCGTCATAGCG-E--K--S--V--T--C--P--T--S--P--E--S--I--S--N--D--S--V--I--A-

TTGATTTTGGCACGAGGCGGTTCCCGTGGCATTCCGCTGAAAAATCTAGCCAAACTCGAC-L--I--L--A--R--G--G--S--R--G--I--P--L--K--N--L--A--K--L--D-

TCGGTGTGGGTTTCAACCGAAGATGATCGGATTGCCCAAGCGGTAGAACGTGACTTCCCG-S--V--W--V--S--T--E--D--D--R--I--A--Q--A--V--E--R--D--F--P-

CACGATCTCGTGAGAGTTCACCTGCGTCCGCCGGAGGTAGCCCAAGACCACACCAGTTCC-M--D--L--V--R--V--M--L--R--P--P--E--V--A--Q--D--M--T--S--S-

ATCGAATCGGTCCGGGAGTTTTTGGATCATCATCCACGGGTGCAGAATGTGGCGCTGGTT-I--E--S--V--R--E--F--L--D--M--M--P--R--V--Q--N--V--A--L--V-

CAGTGCACTTCGCCATTTTTGGGGGTGAGGTATTTGGACGAAGCATTGCAGCGGTTCCAG-Q--C--T--S--P--F--L--G--V--R--Y--L--D--E--A--L--Q--R--F--Q-

GATCGTCAAACGCTGTTGAGTCGAGCGCTTCACACCGCACTCTCCACCGATGGATTTCAC-D--R--Q--T--L--L--S--R--A--L--M--T--A--L--S--T--D--G--F--M-E

GAATAA-E----

TTGGAGATCGATTCGTTGTACGATCTGGAGTTAGCAAGGAAGATCATTGGATCCACAGTA-L--E--I--D--S--L--Y--D--L--E--L--A--R--K--I--I--G--S--T--V-

CTAGAGGGTCGCTTTCAGAACAACAACTGCGAGGTGGTTGTGATTGACGAAAGAGATTCA-L--E--G--R--F--Q--N--N--N--C--E--V--V--V--I--D--E--R--D--S-

GATTGGGACGGAGAGCTTGTTGAGGCGGGGATGTTCTACTTTGCAAGGAGAAAGTTGCTT-D--W--D--G--E--L--V--E--A--G--X--F--Y--F--A--R--R--K--L--L-

GAGAAGGATGGAAGGGTTAATGCGCTGAATTTTGACCCTAGAAAACGTCCCAGGCGTCAA-E--K--D--G--R--V--N--A--L--N--F--D--P--R--K--R--P--R--R--Q-

(c)

(mdash)

pFla

g 2∘

Lec32

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e

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Median

128

0

Cou

nts

100

101

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Fluo

resc

ence

inte

nsity

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(d)

LEC32Lec29 cells Transfected cells WT CHO cells

(e)

Figure 1 AeCSAS functional expression evaluation (a) RT-PCR analysis of AeCSAS and AeST The figure shows the bands obtained withthe internal and external primers of each enzyme using a whole extract of Ae aegypti mosquito Lanes 1-2 AeCSAS (147 bp) and AeST(125 bp) sequences obtained using the internal primers Lanes 3-4 AeCSAS (786 bp) and AeST (1396 bp) complete sequences obtainedwith the external primers Lane 5 Ae actin (298 bp) was used as a housekeeping gene control (b) RT-PCR analysis of AeCSAS and STusing total RNA from five pairs of Ae aegypti SGs (lane 1) and five midguts (lane 2) AeCSAS (147 bp) AeST (125 bp) and actin control(298 bp) (c) cDNA and aa sequences of AeCSAS Identical residues in yellow show multiple alignments with different sequences from otherorganisms (Figure S1) whereas conserved residues are indicated in blue (d) Flow cytometry analysis using LEC29Lec32 untransfected andtransfected cells with AeCSAS which were incubated with MAA lectin to evaluate Sia expression Red isotype control black LEC29Lec32cells transfected with empty p3XFlag-CMV vector (negative control) green untransfected cells in the presence of secondary antibody onlyblue LEC29Lec32 transfectedwithAeCSS cDNA andmagenta wild-typeCHOcells (positive control for the expression of120572-23Sia)Thebarsshow the percentage of fluorescence intensity Approximately 30 of LEC32Lec29-transfected cells expressed Sia (blue bar) compared with100 Sia expression in the positive control CHO cells (magenta bar) (e) Affinocytochemistry and confocal microscopy assays using MAAlectin staining to assess Sia expression Left LEC29Lec32-transfected cells with an empty pFlag vector Center LEC29Lec32-transfected cellswith the AeCSAS pFlag vector Right wild-type CHO positive control transfected with an empty pFlag vector

when we pretreated the SG protein extracts with sialidase(Figure 5(a) lanes 2 and 3)

35 Identification of DENV Attachment Glycoproteins inAe aegypti SGs and Saliva To identify putative sialylatedglycoproteins involved in DENV-SG interactions differentVOPBAs were performed using Ae aegypti SGs and salivaWeobserved thatDENV interactedwith different SGproteinswith approximate molecular weights (MWs) of 115 95 6562 51 37 34 32 17 15 and 9-10 kDa (Figure 5(a) lane 10)

The proteins with MWs from 65 to 9 kDa were also observedin the samples detected with SNA lectin (Figure 5(a) lane 9)To test the possible participation of Sia in DENV-mosquitoprotein interactions we performed a parallel VOBPA assaywhere we pretreated protein extracts from the SGs or salivawith sialidase Interestingly DENV protein binding waspartially or totally abolished in both cases (Figure 5(a) lane11 Figure 5(b) lane 2) It was also interesting that the SGproteins of 95 and 65 kDa which did not interact with SNAlectin (Figure 5(a) lane 9) were not affected in the VOBPA


DAPI SNA lectin MergeSa

livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)

DAPI SNA lectin Merge

Gut

Abdo

men

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gut

(b)

Salivary gland05 IU sialidase 05 IU sialidase 05 IU sialidase

Midgut Head

SNA

lect

inD

API

Aedes aegypti Aedes aegypti Drosophila melanogaster

(c)

Figure 2 Lectin histochemistry of Ae aegypti tissues (a) Results of 120572-26-linked Sia detection inAe aegypti SG midgut and head incubatedwith SNA lectin (1 100) and stained with FITC SG upper panel 60x microscopic magnification lower panel 40x lens The inner box inthe SG-DAPI panel shows the SG region analyzed To identify Sia the midgut and head transverse sections were evaluated with SNA lectin(green) (20x magnification) (b) Results for the 120572-26-linked Sia positive control in D melanogaster abdomen gut and midgut using SNAlectin which are similar to those forAe aegypti tissues (c) SNA staining ofmosquito SG andmidgut pretreatedwith 05 IU sialidase for 30minbefore SNA incubationThe control comprisedDmelanogaster heads pretreatedwith sialidase Blue nuclei stainedwithDAPI Green (FITC)SNA lectin interaction

pretreated with sialidase (Figure 5(a) lane 11) In the saliva-DENV binding assay we observed a protein with a MWof 45 kDa (Figure 5(b) lane 3) which was also presentin the samples with SNA lectin (Figure 5(b) lane 1) butit was eliminated when we used sialidase in the VOPBA(Figure 5(b) lane 2) Thus we propose that the DENV-mosquito SG interaction is at least partially dependent on thepresence of Sia residues We used the sialylated glycoproteinfetuin as a positive control for SNA lectin (Figure 5(a) lanes4 and 12) whereas asialofetuin (Figure 5(a) lanes 5 and 13)and fetuin pretreated with C perfringens sialidase were usedas the negative controls (Figure 5(a) lanes 6 and 14)

36 Identification of Ae aegypti SG and SalivaGlycoproteins byLCESI-MSMS The different DENV-SG and DENV-salivabinding proteins observed in the VOPBAs were identifiedby LCESI-MSMS analysis The identities of the SG andsaliva proteins are shown in Table 1 The DENV-SG bindingproteins were as follows (1) Aedes apyrase which is aprotein that hydrolyzes ATP and ADP to adenosine therebyinhibiting ADP-dependent platelet aggregation (2) Aedessalivary serpin which is an anticoagulant molecule thatinhibits coagulation factor Xa [38] and (3) the Aedes longform of the D7 salivary protein D7 is the most abundantsubfamily of salivary proteins and they are classified as


DENV-SG DAPI

(a)

ConA LcH SNA

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inD

ENV

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PI

(b)

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VD

API

DENV-SG Sialidase Fetuin Free sialic

(c)

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VD

API

Trypsin

5998400

15998400

30998400

(d)

Figure 3 DENV interaction with Ae aegypti SG (a) DENV interaction with Ae aegypti SGs SGs from Ae aegypti were incubated withDENV and stained with anti-DENV E antibody and rhodamine-coupled anti-IgG antibody (b) DENV-SG competence assays using ConALCH and SNA lectins which were added to SG before incubation with DENV The interaction with DENV was blocked when DENV wasincubated in the presence of lectins that recognized SiaWith LCH andConA lectins themagnification = 10x andwith SNA lectin = 20x Scalebar = 10 120583m (c) DENV-SG interaction in the absence or presence of sialidase SGs were untreated or pretreated with C perfringens sialidasefor 30min before adding DENVThe DENV-SG interactions in the presence of Sia competitors fetuin (1mM) and free Sia (200 nM) are alsoshown where the DENV-SG interaction was blocked (d) DENV-SG interaction in SGs pretreated with trypsin for 5 15 or 30min beforeadding DENV There was a decrease in the DENV-SG interaction after 15min and it was lost completely at 30min Scale bar = 10 120583m Bluenuclei stained with DAPI Red DENV stained with an antibody against viral protein E and a secondary antibody coupled to rhodamineGreen (FITC) SNA lectin interaction

odorant pheromone-binding proteins although they alsofunction as scavengers of biogenic amines [39] They alsoinclude (4) the Aedes 30-kDa SG allergen Glycosylatedproteins are associated with allergies [40] Another one ofthe DENV-SG binding proteins is (5) the Aedes putative34 kDa secreted salivary protein which is distributed widelyin mosquito saliva The protein product of the 34 kDa familyhad significant matches with cytoskeletal proteins such asactin and myosin mainly because of the presence of arepeated charged aa [41] Another one of the DENV-SGbinding proteins is (6) the Aedes 145 kDa salivary proteinwhich has an unknown function Another one of the DENV-SG binding proteins is (7) the Aedes short form of the D7salivary protein which can bind biogenic amines such asserotonin histamine and epinephrine [41]The sequestrationof biogenic amines during mosquito feeding is an important

function that inhibits platelet aggregation vasoconstrictionand inflammation Another one of the DENV-SG bindingproteins is (8) theAedesputativeC-type lectin Inmammaliancells two membrane C-type lectins DC-SIGN and L-SIGNinteract with DENV via high-mannose glycans on viralglycoproteins [42] while another C-type lectin the mannosereceptor interacts with the DENV envelope protein whichmay enhance viral attachment to phagocytes [43] It hasalso been demonstrated that the Ae aegypti C-type lectinrecognizes West Nile virus in vivo and in vitro duringcell infection [44] Another one of the DENV-SG bindingproteins is (9) the Aedes beta subunit protein translocationcomplex Silencing of the Drosophila and human orthologgene (Sec61) of the beta subunit protein significantly reducesDENV infections in the S2 cell line and HuH-7 cells [45]Theion masses and the sequences of the SG proteins involved


1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)

Figure 4 SDS-PAGE assay of the glycoproteins from Ae aegypti SG protein extracts (a) Total carbohydrates stained with Pro-Q Emeraldwhere the molecular weights are shown on the right (b) Western blot assay using ConA lectin which binds to glycoproteins that containmannose or glucose residues

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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66

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(kDa)Saliva

(b)

Figure 5 DENV overlay assay with Ae aegypti SG protein extract (SGPE) and Aedes saliva in the presence or absence of C perfringenssialidase (a) DENV-SGPE interactions Lanes 1ndash6 show nitrocellulose membranes stained with Ponceau red Lane 1 MW markers lane 2SGPE lane 3 SGPE pretreated with sialidase lane 4 fetuin glycoprotein lane 5 asialofetuin and lane 6 fetuin pretreated with sialidase Lanes7ndash12 show the blot and overlay assays of SGPE Lane 7 MW markers lane 8 SGPE lane 9 blot of SGPE with SNA lectin lane 10 DENVoverlay with SGPE lane 11 DENV overlay with SGPE pretreated with sialidase lane 12 blot of fetuin glycoprotein with SNA lectin lane 13blot of asialofetuin with SNA lectin and lane 14 blot of SNA lectin with fetuin pretreated with sialidase (b) DENV-saliva interactions Lane1 blot of mosquito saliva with SNA lectin lane 2 DENV overlay with saliva pretreated with sialidase and lane 3 overlay of DENV-salivaproteins

in DENV interactions were evaluated using Glycomod todetermine whether the proteins were putative glycoproteinswith Sia motifs (Supplementary File 1)

The 45-kDa saliva protein that interacts with specificlectins for Sia as well as with DENV is similar to the peptide

ion mass of the protein NCBI gi|157113327 [VectorbaseAAEL006417-RA] which is a putative molecule in the D7family of Ae aegypti It had a 35 match in its primarysequence with a score of 178 and an expected value of64minus13 (119875 lt 005) Based on the analysis of the sequence


Table 1 Identification of DENV-2 binding proteins from Ae aegypti SGs and saliva proteins

Protein name NCBI accession number MW (kDa) Number ofmatched peptides Score Sequence

coverage ()Gel DatabaseSG protein extractApyrase [Aedes aegypti] gi|556272 62820 62691 14 404 19Salivary serpin [Aedes aegypti] gi|94469320 51617 47765 49 712 60D7 protein putative [Aedes aegypti] gi|157113327 37200 38603 46 862 44Long form D7Bclu1 salivary protein [Aedesaegypti] gi|16225992 37200 38579 5 86 15

D7 protein [Aedes aegypti] gi|159557 37200 37005 3 51 9Putative 34 kDa secreted protein [Aedesaegypti] gi|18568296 34833 36154 18 547 33

Putative 34 kDa family secreted salivaryprotein [Aedes aegypti] gi|94468336 34833 35698 20 533 33

30 kDa salivary gland allergen Aed a 3[Aedes aegypti] gi|2114497 32628 27130 37 479 55

Allergen putative [Aedes aegypti] gi|157133926 32628 29529 13 216 31Short form D7Cclu23 salivary protein[Aedes aegypti] gi|16225995 16947 17676 10 150 24

Putative salivary C-type lectin [Aedesaegypti] gi|94468370 16947 17202 5 104 17

Putative 145 kDa salivary protein [Aedesaegypti] gi|94468650 14862 17039 6 117 40

Protein translocation complex beta subunitputative [Aedes aegypti] gi|157138304 9397 10329 2 75 24

SalivaD7 Protein putative [Aedes aegypti] gi|157113327 4523 39173 18 178 35Proteins were identified by LCESI-MSMS analysis after gel trypsin digestion The table shows the protein name the NCBI accession number the theoretical(database) and observed (gel) MWs the number of peptide sequences matched in the MASCOT database the corresponding percentage sequence coverageand the MASCOT score The criteria used for accepting a protein as a valid identification were two or more tryptic peptide matches with the protein sequenceand at least one peptide with 119875 lt 005

of the putative D7 protein from Ae aegypti we identified atransmembrane region between aa residues 7 (phenylalanine)and 24 (leucine) from the amino terminus (Figure S4)Therefore it can be considered as a membrane proteinalthough it has been suggested that members of this familyof proteins are secreted in the salivary glands of variousmosquitoes [46 47] We also noted that the D7 proteincontains potential N-glycosylation sites specifically in theregion of aas 278ndash284 (Supplementary File 1) There weretwo possible combinations of carbohydrates involving Siathe first was combined with hexose and the second with N-acetylglucosamine or N-acetylgalactosamine We evaluatedthe potential Sia-glycosylation sites some of which havelittle differences in terms of the ionic masses obtained withMALDI-ToF (experimental mass) the theoretical mass of theglycopeptides and the carbohydrate mass In addition weonly considered differences of lt005Da and three peptideregions in the D7 protein had these characteristics Betweenresidues 35ndash39 there were two possible combinations ofO-linked glycosylation via the hydroxyl groups of serineand threonine the first combination involved the bindingof Sia to two molecules of N-acetylglucosamine or N-acetylgalactosamine and the second involved a combina-tion with hexose NeuAc and ketodeoxynonulosonic acid

The second peptide with the potential to be O-glycosylatedwas in the region of aas 285ndash290 where a threonine residuecould be linked to pentose N-acetylglucosamine or N-acetylgalactosamine and Sia residues Finally there wasa serine residue in the region of aas 311ndash316 where thedifference between the experimental mass and theoreticalmass was only 0019Da Therefore it is possible that a Siaresidue linked to a deoxyhexose occurs in this region

37 DENV Infection of Mammalian Cells in the Presence ofAe aegypti SG Protein Extracts It is known that Ae aegyptisaliva enhances West Nile and Cache Valley virus infectionsbut it is unknown whether Aedes saliva can modulate DENVinfections [6] Based on our detection of interactions betweenDENV and salivary glycoproteins we evaluated the possibleparticipation of the Ae aegypti SG protein extract in themodulation of DENV infection in different mammalian celllines (LLCMK2 andCHOWT) using aDENV internalizationassay in the presence or absence of SG extracts We foundthat DENV infection was enhanced in the presence of SGextract in both mammalian cell lines (Figure 6(a)) CHOcells appeared to be more permissive (fourfold enhancementFigure 6(a) lane 7) than LLCMK2 (twofold enhancementFigure 6(a) lane 3)We pretreated the SGprotein extract with


lowast

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Figure 6 DENV-mammalian cells internalization assay (a) DENV internalization by LLC-MK2 and CHO cells The plot shows theinternalization of [35S]-methionine-radiolabeled DENV by LLC-MK2 and CHO cells in the absence (lanes 2 and 6) and presence (lanes3 and 7) of Ae aegypti SG protein extract (SGH) and in the presence of SGH pretreated with sialidase before DENV incubation (lanes 4 and8) DENV was mixed with SGH (from 80 SGs) which was pretreated or untreated with sialidase before infecting mammalian cells with theDENV-SGH mixture In the plot the 119910-axis shows the counts per min of internalized DENV lowast119875 lt 005 (b) DENV internalization by CHOcells in the presence of different amounts of SGH The plot shows that DENV internalization was enhanced by the presence of the proteinextract from five SGs which was dose dependent

sialidase before the internalization assay to evaluate the pos-sible participation of Sia during DENV cell internalizationand we observed the effect on DENV internalization whichwas reduced in sialidase-pretreated samples (Figure 6(a)lanes 4 and 8) The internalization of DENV in CHO cellsin the presence of different amounts of SG protein extractwas dose dependent as shown in Figure 5(b) These resultssupport a general hypothesis that molecules in mosquitosaliva and secretory SG proteins can potentiate pathogen-host transmission and that Sia residues play a role duringDENV internalization in mammalian cells

4 Discussion

Sialylation is a biologically important modification of glyco-conjugates which is observed mainly in the deuterostomelineage However the occurrence of this process in pro-tostomes is less clear [19] Using the available Ae aegyptigenome database we identified two putative genes encodingenzymes (AeCSAS and AeST) implicated in the Ae aegyptisialylation pathway The cDNA of AeCSAS was amplifiedcloned and functionally evaluated by the complementationof CSAS-deficient LEC29Lec32 CHO cells Sia moieties werepresent at the cell surface in AeCSAS-transfected CHO

LEC29Lec32 cells The identification of a functional Siasynthase in Ae aegypti indicates that Aedesmosquitoes havethe biosynthetic capacity for endogenous Sia productionOur data are consistent with previous studies [12ndash16] of theexpression of a functional D melanogaster CSAS and thepresence of 120572-26-linked Sia moieties in D melanogaster Siais distributed widely in nature at the nonreducing termini ofglycoproteins glycolipids or secreted glycoconjugates andit may be attached to different acceptors via 120572-23 120572-26or 120572-28-linkages which are determined by the specificityof different STs [48] In this study we demonstrated thepresence of Ae aegypti ST cDNAs in different Ae aegyptitissues (Figures 1(a) and 1(b)) and observed the presence of120572-26-linked Sia moieties (in a lectin binding assay) at thetissue level These data are consistent with a report where itwas shown that arthropods STs including Ae aegypti ST areassociatedwith the ST6Gal ST family which is orthologous tothe common ancestral gene that was present before the splitof ST6Gal I and ST6Gal II in vertebrates [34]

To our knowledge this is the first report of the presenceof Sia glycans in Ae aegypti tissues The type of Sia linkagealso plays a key role in the specific recognition of differentviruses because 120572-23- or 120572-26-specificity could define thecell and host tropism [49] For example human influenza


A virus hemagglutinin binds primarily to Neu5Ac1205722-6Galstructures whereas avian influenza virus binds specifically toNeu5Ac1205722-3Gal [50] This specificity limits the cell tropismand viral host range significantly The participation of 120572-26-Sia structures during early DENV-vector interactions mayhave key roles in DENV infection host tropism and viralpathogenesis

It was reported that Anopheles salivary glands containseveral glycoconjugates in the surface which are criticalfor recognition of different pathogens [51ndash53] Perroneet al [54] suggested that the salivary gland carbohydratecomplexity reflects the functional diversity of this tissueBy lectin-binding assay the authors detected the presenceof 120572-D-mannose 120572-D-N-acetyl-galactosamine 120573-D-gal-(13) N-acetyl-galactosamine 120573-D-galactose N-acetyl-galactosamine 120572-L-fucose and 120573-N acetyl-glucosamineLikewise different oligosaccharide structures such asMan3GlcNAc2 Man3 (Fuc) 1-2GlcNAc2 were detected[55] Recently Francischetti et al [56] demonstrated thepresence of sulfated glycans in the salivary gland ofAnophelesgambiae Because of the glycan complexity in the vectorsalivary glands and in order to ensure that sialic aciddetection in Aedes aegypti mosquito tissues was specificthe role of Sia in DENV-SG binding was evaluated by aDENV-SG competition assay using free sialic acid and alsothe sialylated glycoprotein fetuin We observed that theDENV-SG interaction decreased in the presence of fetuinand it was lost in the presence of free sialic acid (Figure 3(c)and supplementary Figure 5(B)ndash5(E)) In the same way weobserved in a hemagglutination assay of dengue virus withsialylated red blood cell (chicken erythrocytes) an inhibitoryeffect in presence of free sialic acid Moreover the Siaparticipation in DENV-sialic acid interaction was confirmedby the loss of hemmagglutination activity in the presence ofdesialylated erythrocytes (Supplementary Table 1)

DENV cellular infection is a multistep process thatinvolves different molecules some of them present in Aedestissues like the laminin receptor the tubulin like proteinHSP90 protein unknown proteins of Mw 35 40ndash45 48 74and 80KDa and several detergent-soluble proteins of salivaryglands withMw 35ndash80KDa [57] However neither evaluatedthe possible participation of Sia glycoconjugates [58 59]and the occurrence and participation of Sia in interactionsamongmosquito tissues andDENVhave not been consideredpreviously

However the participation of Sia in Plasmodium galli-naceum ookinetes-midgut interactions has been documentedpreviously Zieler et al [60] reported that the chemicalmodification of the midguts from Ae aegypti mosquito witha periodate concentration of lt1mmol inhibit the adhesionof ookinetes in the midguts and they also found that freeN-acetylneuraminic acid competed for ookinete binding tomidguts Interestingly Barreau et al [61] found that wheat-germ agglutinin (WGA) lectin which binds residues of N-acetylglucosamine blocks the interaction between Plasmod-ium gallinaceum sporozoites and the surface of Ae aegyptiSGs WGA is a Triticum vulgaris lectin that specifically

recognizes N-acetylglucosamine residues but it also hasregions that interact with Sia residues These reports suggestthe possible participation (and presence) of sialic acid inthe interactions between mosquito tissue and PlasmodiumColpitts et al [9] reported that Sia residues are importantfor the recognition of DENV in mammalian (Vero andLLC-MK2) cells and a large number of DENV bindingmolecules are known [58 62ndash66] However there have beenno evaluations of the possible role during DENV-vector-hosttransmission

In the present study we found that a sialylated salivaglycoprotein (45 kDa Figure 5(b) lane 1) of Ae aegypti formscomplexes with DENV This protein belongs to the D7proteins family and is secreted in the saliva [21] thus it couldbe implicated in DENV host transmission The modulationof DENV infection in different mammalian cells by Aedessalivary extracts and the observation that desialylated salivaryproteins decrease DENV internalization highlight the keyroles played by sialylated molecules during DENV vector-host interactions Several studies have demonstrated theeffects of arthropod saliva on vertebrate responses in a widerange of disease models using various hosts arboviruses andmosquito species [5 6 67 68] In all cases an increase in virustransmission modification of host susceptibility or diseaseprogression were observed The enhancement of infectionas a result of SG extracts is attributed to the modulationof host immune response reduction of T-lymphocytes andantiviral activity [69] In the current study we detectedenhanced DENV internalization in presence of Aedes SGextracts but the virus internalization decreased when thesalivary proteins were pretreated with sialidase In agreementwith our results Surasombatpattana et al [70 71] observedenhanced DENV infection of human keratinocytes in thepresence of SG extracts Recently Conway et al [7] reportedthat theAedes aegypti saliva serine protease activity enhancesdissemination of DENV into the mammalian host althoughthe role of Sia was not considered Identification of moleculesthat mediate infectivity enhancement will allow for theproduction of vector-based vaccines and therapeutics thatwill target arthropod saliva components and interfere withviral transmission as is exemplified by antimaxadilan (MAX)and anti-SP15 vaccines [72 73] These data may representa general property for other vector-borne pathogens as isthe case of Plasmodium The knowledge of early DENV-host interactions could lead to a better understanding ofviral tropism and pathogenesis and provide information forthe development of new strategies for the control of DENVtransmission

To our knowledge this is the first report of the partic-ipation of Sia structures during early interactions betweenDENV and Ae aegyptimosquito tissues

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The authors thank Dr Jorge Guevara of the National Instituteof Neurology for his support with the lectin immunohis-tochemistry assays and Gerardo Hurtado and Dr VictoriaPando of the INSP for the initial D7MALDI-TOF assayTheyalso thank Miguel Tapia Rodrıguez for his excellent supportof the confocal assay And they also thank Dr P Stanleyfrom the Albert Einstein College of Medicine at YeshivaUniversity NY for kindly providing LEC29Lec32 cells JorgeCime was a PhD student in the Programa de Doctorado enCiencias Biomedicas Universidad Nacional Automoma deMexico and received a scholarship from Consejo Nacionalde Ciemcia Tecnologia (CONACYT)MexicoThis study wassupported by CONACYT and PAPIIT-UNAM grants

References

[1] World Health Organization (WHO) Dengue Guidelines forDiagnosis Treatment Prevention and Control WHO GenevaSwitzerland 2009 httpwwwwhointtopicsdengueen

[2] D J Gubler ldquoThe changing epidemiology of yellow fever anddengue 1900 to 2003 full circlerdquo Comparative ImmunologyMicrobiology amp Infectious Diseases vol 27 no 5 pp 319ndash3302004

[3] S B Halstead ldquoDengue virus-mosquito interactionsrdquo AnnualReview of Entomology vol 53 pp 273ndash291 2008

[4] J R Anderson and R Rico-Hesse ldquoAedes aegypti vectorialcapacity is determined by the infecting genotype of denguevirusrdquoThe American Journal of Tropical Medicine and Hygienevol 75 no 5 pp 886ndash892 2006

[5] R G Titus J V Bishop and J S Mejia ldquoThe immunomod-ulatory factors of arthropod saliva and the potential for thesefactors to serve as vaccine targets to prevent pathogen trans-missionrdquo Parasite Immunology vol 28 no 4 pp 131ndash141 2006

[6] B S Schneider and S Higgs ldquoThe enhancement of arbovirustransmission and disease by mosquito saliva is associated withmodulation of the host immune responserdquo Transactions of theRoyal Society of Tropical Medicine and Hygiene vol 102 no 5pp 400ndash408 2008

[7] M J Conway A M Watson T M Colpitts et al ldquoMosquitosaliva serine protease enhances dissemination of dengue virusinto the mammalian hostrdquo Journal of Virology vol 88 no 1 pp164ndash175 2014

[8] SThangamani and SKWikel ldquoDifferential expression ofAedesaegypti salivary transcriptome upon blood feedingrdquo Parasitesand Vectors vol 2 no 1 article 34 2009

[9] TM Colpitts J Cox D L Vanlandingham et al ldquoAlterations inthe aedes aegypti transcriptome during infection with west niledengue and yellow fever virusesrdquo PLoS Pathogens vol 7 no 9Article ID e1002189 2011

[10] B K Thaisomboonsuk E T Clayson S Pantuwatana DW Vaughn and T P Endy ldquoCharacterization of dengue-2virus binding to surfaces of mammalian and insect cellsrdquo TheAmerican Journal of Tropical Medicine and Hygiene vol 72 no4 pp 375ndash383 2005

[11] V Stollar ldquoTogaviruses in cultured arthropod cellsrdquo in TheTogaviruses Biology Structure Replication R W SchlesingerEd pp 76ndash84 Academic Press New York NY USA 1980

[12] J Roth A Kempf G Reuter R Schauer and W J GehringldquoOccurrence of sialic acids inDrosophila melanogasterrdquo Sciencevol 256 no 5057 pp 673ndash675 1992

[13] K Kim SM Lawrence J Park et al ldquoExpression of a functionalDrosophila melanogaster N-acetylneuraminic acid (Neu5Ac)phosphate synthase gene evidence for endogenous sialic acidbiosynthetic ability in insectsrdquo Glycobiology vol 12 no 2 pp73ndash83 2002

[14] K Koles K D Irvine and V M Panin ldquoFunctional characteri-zation of Drosophila sialyltransferaserdquoThe Journal of BiologicalChemistry vol 279 no 6 pp 4346ndash4357 2004

[15] K Aoki M Perlman J-M Lim R Cantu L Wells and MTiemeyer ldquoDynamic developmental elaboration of N-linkedglycan complexity in theDrosophila melanogaster embryordquoTheJournal of Biological Chemistry vol 282 no 12 pp 9127ndash91422007

[16] K Viswanathan N Tomiya J Park et al ldquoExpression of afunctionalDrosophilamelanogaster CMP-sialic acid synthetaseDifferential localization of the drosophila and human enzymesrdquoThe Journal of Biological Chemistry vol 281 no 23 pp 15929ndash15940 2006

[17] E Repnikova K Koles M Nakamura et al ldquoSialyltransferaseregulates nervous system function in Drosophilardquo The Journalof Neuroscience vol 30 no 18 pp 6466ndash6476 2010

[18] R Islam M Nakamura H Scott et al ldquoThe role of Drosophilacytidine monophosphate-sialic acid synthetase in the nervoussystemrdquo Journal of Neuroscience vol 33 no 30 pp 12306ndash123152013

[19] A Varki ldquoGlycan-based interactions involving vertebrate sialic-acid-recognizing proteinsrdquo Nature vol 446 no 7139 pp 1023ndash1029 2007

[20] C Cabello-Gutierrez M E Manjarrez-Zavala A Huerta-Zepeda et al ldquoModification of the cytoprotective protein Cpathway during Dengue virus infection of human endothelialvascular cellsrdquo Thrombosis and Haemostasis vol 101 no 5 pp916ndash928 2009

[21] L Almeras A Fontaine M Belghazi et al ldquoSalivary glandprotein repertoire from Aedes aegypti mosquitoesrdquo Vector-Borne and Zoonotic Diseases vol 10 no 4 pp 391ndash402 2010

[22] J P Kamerling G J Gerwig J F Vliegenthart and J RClamp ldquoCharacterization by gas-liquid chromatography-massspectrometry and proton-magnetic-resonance spectroscopy ofpertrimethylsilyl methyl glycosides obtained in the methanoly-sis of glycoproteins and glycopeptidesrdquoBiochemical Journal vol151 no 3 pp 491ndash495 1975

[23] G Reuter and R Schauer ldquoDetermination of sialic acidsrdquoMethods in Enzymology vol 230 pp 168ndash199 1994

[24] S Hara M Yamaguchi Y Takemori K Furuhata H Oguraand M Nakamura ldquoDetermination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometrichigh-performance liquid chromatographyrdquoAnalytical Biochem-istry vol 179 no 1 pp 162ndash166 1989

[25] S S Twining ldquoFluorescein isothiocyanate-labeled casein assayfor proteolytic enzymesrdquoAnalytical Biochemistry vol 143 no 1pp 30ndash34 1984

[26] E Tian L Zhang and K G T Hagen ldquoFluorescent lectinstaining of drosophila embryos and tissues to detect the spatialdistribution of glycans during developmentrdquoMethods inMolec-ular Biology vol 1022 pp 99ndash105 2013

[27] J S Salas-Benito and R M del Angel ldquoIdentification of twosurface proteins fromC636 cells that bind dengue type 4 virusrdquoJournal of Virology vol 71 no 10 pp 7246ndash7252 1997


[28] M Kinter and N E Sherman ldquoThe preparation of proteindigests for mass spectrometric sequencing experimentsrdquo inProtein Sequencing and Identification Using Tandem Mass Spec-trometry Wiley-Interscience New York NY USA 2000

[29] C A Cooper E Gasteiger and N H Packer ldquoGlycoModmdashasoftware tool for determining glycosylation compositions frommass spectrometric datardquo Proteomics vol 1 no 2 pp 340ndash3492001

[30] R S Goldsmith ldquoAssay of dengue virus in monkey kidneycells by detection of hemagglutinin in the culture mediumrdquoAmerican Journal of Epidemiology vol 84 no 2 pp 343ndash3511966

[31] J Casals and L V Brown ldquoHemagglutination with arthropod-borne virusesrdquo The Journal of Experimental Medicine vol 99no 5 pp 429ndash449 1954

[32] K Sano and H Ogawa ldquoHemmagglutination (inhibition)assayrdquo in Lectin Methods and Protocols Methods in MolecularBiology vol 1200 pp 47ndash52 2014

[33] R Schauer G V Srinivasan B Coddeville J-P Zanetta andY Guerardel ldquoLow incidence of N-glycolylneuraminic acid inbirds and reptiles and its absence in the platypusrdquo CarbohydrateResearch vol 344 no 12 pp 1494ndash1500 2009

[34] K Koles E Repnikova G Pavlova L I Korochkin andV M Panin ldquoSialylation in protostomes a perspective fromDrosophila genetics and biochemistryrdquo Glycoconjugate Journalvol 26 no 3 pp 313ndash324 2009

[35] B Potvin T S Raju and P Stanley ldquolec32 is a new mutationin Chinese hamster ovary cells that essentially abrogates CMP-N-acetylneuraminic acid synthetase activityrdquo The Journal ofBiological Chemistry vol 270 no 51 pp 30415ndash30421 1995

[36] P Stanley T Shantha Raju and M Bhaumik ldquoCHO cells pro-vide access to novel N-glycans and developmentally regulatedglycosyltransferasesrdquo Glycobiology vol 6 no 7 pp 695ndash6991996

[37] D Petit A-M Mir J-M Petit et al ldquoMolecular phylogeny andfunctional genomics of 120573-galactoside 12057226-sialyltransferasesthat explain ubiquitous expression of st6gal1 gene in amniotesrdquoThe Journal of Biological Chemistry vol 285 no 49 pp 38399ndash38414 2010

[38] K R Stark and A A James ldquoIsolation and characterizationof the gene encoding a novel factor Xa-directed anticoagulantfrom the yellow fever mosquito Aedes aegyptirdquo The Journal ofBiological Chemistry vol 273 no 33 pp 20802ndash20809 1998

[39] E Calvo B J Mans J F Andersen and J M C RibeiroldquoFunction and evolution of a mosquito salivary protein familyrdquoThe Journal of Biological Chemistry vol 281 no 4 pp 1935ndash1942 2006

[40] F E R Simons and Z Peng ldquoMosquito allergy recombinantmosquito salivary antigens for new diagnostic testsrdquo Interna-tional Archives of Allergy and Immunology vol 124 no 1ndash3 pp403ndash405 2001

[41] J M C Ribeiro B Arca F Lombardo et al ldquoAn annotatedcatalogue of salivary gland transcripts in the adult femalemosquito Aedes aegyptirdquo BMC Genomics vol 8 article 6 2007

[42] W B Klimstra E M Nangle M S Smith A D Yurochko andK D Ryman ldquoDC-SIGN and L-SIGN can act as attachmentreceptors for alphaviruses and distinguish between mosquitocell and mammalian cell-derived virusesrdquo Journal of Virologyvol 22 no 77 pp 12022ndash12032 2003

[43] J L Miller B J M de Wet L Martinez-Pomares et alldquoThe mannose receptor mediates dengue virus infection ofmacrophagesrdquo PLoS Pathogens vol 4 no 2 article e17 2008

[44] G Cheng J Cox P Wang et al ldquoA C-type lectin collaborateswith a CD45 phosphatase homologue to facilitate West Nilevirus infection of mosquitoesrdquo Cell vol 142 no 5 pp 714ndash7252010

[45] OM Sessions N J Barrows J A Souza-Neto et al ldquoDiscoveryof insect and human dengue virus host factorsrdquoNature vol 458no 7241 pp 1047ndash1050 2009

[46] B Arca F Lombardo M de Lara Capurro et al ldquoTrappingcDNAs encoding secreted proteins from the salivary glandsof the malaria vector Anopheles gambiaerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 4 pp 1516ndash1521 1999

[47] B Arca F Lombardo A Lanfrancotti et al ldquoA cluster of fourD7-related genes is expressed in the salivary glands of theAfrican malaria vector Anopheles gambiaerdquo Insect MolecularBiology vol 11 no 1 pp 47ndash55 2002

[48] A Harduin-Lapers ldquoComprehensive analysis of sialyltrans-ferases in vertebrate genomesrdquo Glycobiology Insights vol 2 pp29ndash61 2010

[49] F Lehmann E Tiralongo and J Tiralongo ldquoSialic acid-specificlectins occurrence specificity and functionrdquo Cellular andMolecular Life Sciences vol 63 no 12 pp 1331ndash1354 2006

[50] Y Suzuki T Ito T Suzuki et al ldquoSialic acid species as adeterminant of the host range of influenza A virusesrdquo Journalof Virology vol 74 no 24 pp 11825ndash11831 2000

[51] J D G Brennan M Kent R Dhar H Fujioka and N KumarldquoAnopheles gambiae salivary gland proteins as putative targetsfor blocking transmission of malaria parasitesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 97 no 25 pp 13859ndash13864 2000

[52] A A James ldquoBlocking malaria parasite invasion of mosquitosalivary glandsrdquo The Journal of Experimental Biology vol 206no 21 pp 3817ndash3821 2003

[53] A K Ghosh and M Jacobs-Lorena ldquoPlasmodium sporozoiteinvasion of the mosquito salivary glandrdquo Current Opinion inMicrobiology vol 12 no 4 pp 394ndash400 2009

[54] J B Perrone J DeMaio and A Spielman ldquoRegions of mosquitosalivary glands distinguished by surface lectin-binding charac-teristicsrdquo Insect Biochemistry vol 16 no 2 pp 313ndash318 1986

[55] J S Li C J Vavricka B M Christensen and J Li ldquoProteomicanalysis of N-glycosylation in mosquito dopachrome conver-sion enzymerdquo Proteomics vol 7 no 15 pp 2557ndash2569 2007

[56] I M Francischetti D Ma J F Andersen and J M RibeiroldquoEvidence for a lectin specific for sulfated glycansin the salivarygland of the malaria vector Anopheles gambiaerdquo PLoS ONEvol 10 no 9 Article ID e107295 2014

[57] K I P J Hidari and T Suzuki ldquoDengue virus receptorrdquo TropicalMedicine and Health vol 39 no 4 pp 37ndash43 2011

[58] A Cabrera-Hernandez and D R Smith ldquoMammalian denguevirus receptorsrdquo Dengue Bulletin vol 29 pp 119ndash133 2005

[59] D R Smith ldquoAn update on mosquito cell expressed denguevirus receptor proteinsrdquo Insect Molecular Biology vol 21 no 1pp 1ndash7 2012

[60] H Zieler J P Nawrocki and M Shahabuddin ldquoPlasmod-ium gallinaceum ookinetes adhere specifically to the midgutepithelium of Aedes aegypti by interaction with a carbohydrateligandrdquoThe Journal of Experimental Biology vol 202 no 5 pp485ndash495 1999


[61] C BarreauM Touray P F Pimenta L HMiller and K D Ver-nick ldquoPlasmodium gallinaceum sporozoite invasion of Aedesaegypti salivary glands in inhibited by anti-gland antibodies andby lectinsrdquo Experimental Parasitology vol 81 no 3 pp 332ndash3431995

[62] M Y Mendoza J S Salas-Benito H Lanz-Mendoza SHernandez-Martinez and R M del Angel ldquoA putative receptorfor dengue virus in mosquito tissues localization of a 45-KDAglycoproteinrdquo The American Journal of Tropical Medicine andHygiene vol 67 no 1 pp 76ndash84 2002

[63] R F Mercado-Curiel H A Esquinca-Aviles R Tovar ADıaz-Badillo M Camacho-Nuez and M de Lourdes MunozldquoThe four serotypes of dengue recognize the same putativereceptors inAedes aegyptimidgut andAe albopictus cellsrdquoBMCMicrobiology vol 6 article 85 2006

[64] R F Mercado-Curiel W C Black IV and M D L Muoz ldquoAdengue receptor as possible genetic marker of vector compe-tence in Aedes aegyptirdquo BMC Microbiology vol 8 article 1182008

[65] V-M Cao-Lormeau ldquoDengue viruses binding proteins fromAedes aegypti and Aedes polynesiensis salivary glandsrdquo VirologyJournal vol 6 article 35 2009

[66] M D L Munoz G Limon-Camacho R Tovar A Diaz-Badillo G Mendoza-Hernandez and W C Black ldquoProteomicidentification of dengue virus binding proteins in Aedes aegyptimosquitoes and Aedes albopictus cellsrdquo BioMed Research Inter-national vol 2013 Article ID 875958 11 pages 2013

[67] R G Titus and J M C Ribeiro ldquoSalivary gland lysates from thesand fly Lutzomyia longipalpis enhance leishmania infectivityrdquoScience vol 239 no 4845 pp 1306ndash1308 1988

[68] L M Styer K A Kent R G Albright C J Bennett L DKramer and K A Bernard ldquoMosquitoes inoculate high dosesof West Nile virus as they probe and feed on live hostsrdquo PLoSPathogens vol 3 no 9 article e132 2007

[69] B S Schneider L Soong L L Coffey H L Stevenson CE McGee and S Higgs ldquoAedes aegypti saliva alters leukocyterecruitment and cytokine signaling by antigen-presenting cellsduringWestNile virus infectionrdquoPLoSONE vol 5 no 7 ArticleID e11704 2010

[70] P Surasombatpattana S Patramool N Luplertlop H Yssel andD Misse ldquoAedes aegypti saliva enhances dengue virus infec-tion of human keratinocytes by suppressing innate immuneresponsesrdquoThe Journal of InvestigativeDermatology vol 132 no8 pp 2103ndash2105 2012

[71] P Surasombatpattana P Ekchariyawat R Hamel et al ldquoAedesaegypti saliva contains a prominent 34-kDa protein thatstrongly enhances dengue virus replication in human ker-atinocytesrdquo Journal of Investigative Dermatology vol 134 no 1pp 281ndash284 2014

[72] R V Morris C B Shoemaker J R David G C Lanzaroand R G Titus ldquoSandfly maxadilan exacerbates infection withLeishmania major and vaccinating against it protects againstL major infectionrdquo Journal of Immunology vol 167 no 9 pp5226ndash5230 2001

[73] J G Valenzuela Y Belkaid M K Garfield et al ldquoTowarda defined anti-Leishmania vaccine targeting vector antigenscharacterization of a protective salivary proteinrdquo Journal ofExperimental Medicine vol 194 no 3 pp 331ndash342 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


The female mosquito acquires DENV from an infectedperson during blood feeding The virus undergoes its firstreplication cycle in the mosquito midgut before spreadinginto the hemocoel and finally infecting the salivary glands(SGs) The transfer of infectious saliva into a human host(during a new blood feeding) is a key event during the DENVtransmission cycle [4 5]Thus it is very important to identifythemolecules involved in the DENV-SG relationship becausemosquito saliva is rich in glycoproteins that participate indifferent host responses (platelet activation swelling itchingand inflammation) as well as the binding and transportof vector-borne pathogens to host tissues thereby allowingpathogens to infect and evade the host immune response[5] In an ample range of disease models including vari-ous hosts mosquito species and arthropod-borne virusesmosquito saliva andor mosquito feeding are associated witha potentiation of the arbovirus (arthropod-borne) infectionHost infection via vector saliva leads to an increase in viraltransmission host susceptibility disease progression andmortality [6] The potential for mosquitoes to influence thecourse of West Nile virus (WNV) disease was investigatedby assessing pathogenesis in the presence or absence ofmosquito saliva [6] Likewise in vitro and in vivo modelsof saliva-mediated enhancement of DENV infectivity havebeen reported [7] but it is uncertain whether Aedes salivaglycosylatedmolecules contributes to DENV tissue infectionThe Aedes sialome includes 136 putative secretory proteinswhich couldmodify host responses [8]DuringDENV-vectorinfection the main genes upregulated in Ae aegypti arerelated to carbohydrate expression [9] but the roles of glycansin vector competence are currently unknown In additionit is known that certain glycosidases affect the binding ofDENV to mammalian (green monkey kidney and Vero) andmosquito (C636 and AP61) cell surfaces [10] Previously itwas reported that 120573-glucosidase sialidase and heparinasereduce DENV attachment to mammalian cells but not toinsect cells [10] and the inability of sialidase to affect DENVbinding to insect cells is associated with a lack of mosquitosialyltransferase (ST) which is capable of transferring sialicacid (Sia) residues to mosquito glycoproteins [11] Moreoverthe occurrence of Sia in mosquito tissues is also unknownHowever the genetic and biochemical capacity for sialylationin Drosophila melanogaster supports a hypothesis that insectsialylation is a specialized and developmentally regulatedprocess in insects [12ndash16] This process is involved in theregulation of neural transmission in the nervous system ofD melanogaster [17 18] It is well known that sialylatedglycoproteinsmodulatemany important biological processesincluding cellular andmolecular recognition subcellular andcellular trafficking intercellular adhesion and signaling andmicrobial attachment among others [19] In the presentstudy we detected the presence of a functional cytidinemonophosphate- (CMP-)Sia synthase (CSAS) in Ae aegyptiand we also demonstrated that DENV recognizes 120572-26-linked Sia structures on the surface ofmosquito tissues whichmay play key roles during early DENV-vector interactionsFurthermore we found that DENV is capable of interactingwith secretory Sia-glycoproteins which may be involved in

successful DENV-host tissue transmission To our knowl-edge these are the first demonstrations of the functionalexpression of anAedesCSAS and the presence of Sia moietiesin mosquito tissues which may have important biologicalconsequences for DENV-vector competence Knowledge ofspecific early DENV-mosquito interactions could facilitatea better understanding of viral tropism and pathogenesisto allow the development of new effective strategies for thecontrol of DENV transmission as well as the improvementof antiviral agents and vaccines

2 Materials and Methods

21 DENV Propagation and Titration DENV New GuineaC strain serotype 2 (DENV-2 kindly donated by Dr DuaneGubler CDC Fort Collins CO USA) was propagated inC636 cells which were grown at 28∘C in supplementedminimal essential medium (MEM) Confluent monolayerswere infected for 2 h at a multiplicity of infection (MOI)of 1 and incubated for 5ndash7 days at 28∘C in a 5 CO

2

atmosphere until cytopathic effects were observed beforetitrating in a lytic plaque assay using LLC-MK2 cells asdescribed previously [20] The virus titer was expressed asplaque-forming units (pfu) per milliliter

22 Ae aegypti Maintenance Salivary Glands Midgut Iso-lation and Tissue Extracts Female Ae aegypti mosquitoeswere cultured in an insectarium at the Center for Infec-tious Disease Research (CISEI-INSP) Mexico The SGs andmidguts of female mosquitoes (at least three days old and fedonly with water) were dissected using a microneedle placedin sterile tubes in groups of 20 pairs with 20120583L of phosphate-buffered saline (PBS) and kept at minus75∘C The tissues werelysed during five freeze-thaw cycles using liquid nitrogenand sonicated (ultrasonic 8849-00 Cole-Parmer IL USA)for 10min before centrifugation at 3500 rpm to obtain tissueextracts The protein concentration was determined usinga micro-BCA (bicinchoninic acid) assay (Pierce USA) at562 nm with a spectrophotometer (Multiskan Ascent 354Thermo Labsystem UK)

23 Ae aegypti Saliva Collection Ae aegypti saliva wascollected as described by Almeras et al [21] with a smallnumber of modifications Female mosquitoes were sedatedfor 1min at 4∘C and the proboscis of each mosquito wasplaced in a plastic pipette tip containing mineral oil After1 h salivation at room temperature (RT) the liquid wascollected from the tip and the saliva from 20 mosquitoeswas pooled before centrifugation at 10000 rpm The proteinconcentration was estimated using a micro-BCA assay

24 Carbohydrate Determination in Ae aegypti SalivaryGlands The salivary glands of femaleAe aegyptimosquitoeswere dissected as described above and the SG monosac-charides were analyzed according to Kamerling et al[22] by GCMS as trimethylsilyl methyl glycosides (bythe Structural and Functional Glycobiology Unit of theUniversity of Sciences and Technologies of Lille France)Briefly dry samples were methanolized in methanolHCl

































3 Results










1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

147bp

125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260










GAATAA-E----





(c)

(mdash)

pFla

g 2∘

Lec32

CMPA

e

WT

Median

128

0

Cou

nts

100

101

102

103

104

100

80

60

40

20

Fluo

resc

ence

inte

nsity

()

(d)


(e)







livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































3 Results










1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

147bp

125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260










GAATAA-E----





(c)

(mdash)

pFla

g 2∘

Lec32

CMPA

e

WT

Median

128

0

Cou

nts

100

101

102

103

104

100

80

60

40

20

Fluo

resc

ence

inte

nsity

()

(d)


(e)







livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























3 Results










1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

147bp

125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260










GAATAA-E----





(c)

(mdash)

pFla

g 2∘

Lec32

CMPA

e

WT

Median

128

0

Cou

nts

100

101

102

103

104

100

80

60

40

20

Fluo

resc

ence

inte

nsity

()

(d)


(e)







livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














3 Results










1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

147bp

125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260










GAATAA-E----





(c)

(mdash)

pFla

g 2∘

Lec32

CMPA

e

WT

Median

128

0

Cou

nts

100

101

102

103

104

100

80

60

40

20

Fluo

resc

ence

inte

nsity

()

(d)


(e)







livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

147bp

125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260










GAATAA-E----





(c)

(mdash)

pFla

g 2∘

Lec32

CMPA

e

WT

Median

128

0

Cou

nts

100

101

102

103

104

100

80

60

40

20

Fluo

resc

ence

inte

nsity

()

(d)


(e)







livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5

1396bp786bp

298bp147bp125 bp

(a)

1 2

298bp

147bp

125 bp

Actin

CMP-NeuAcsynthase

Sialyltransferase

(b)

1

1

61

21

121

41

181

61

241

81

301

101

361

121

421

141

481

161

541

181

601

201

661

221

721

241

781

260










GAATAA-E----





(c)

(mdash)

pFla

g 2∘

Lec32

CMPA

e

WT

Median

128

0

Cou

nts

100

101

102

103

104

100

80

60

40

20

Fluo

resc

ence

inte

nsity

()

(d)


(e)







livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



livar

y gl

and

Saliv

ary

glan

dM

idgu

tH

ead

(a)


Gut

Abdo

men

Mid

gut

(b)


Midgut Head

SNA

lect

inD

API


(c)





DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DENV-SG DAPI

(a)

ConA LcH SNA

Lect

inD

ENV

DA

PI

(b)

DEN

VD

API


(c)

DEN

VD

API

Trypsin

5998400

15998400

30998400

(d)





1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 (kDa)

(a)

ConA

180

116

97

66

45

29

21

(b)


1 2 3 4 5 6 7 8 9 10 11 12 13 14

SGPE

11697

66

45

31

21


66

45

31

21

14

11595

6562

51

323437

17159

(a)

1 2 3

45

(kDa)Saliva

(b)





















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast

lowast

lowast

lowast

1 2 3 4 5 6 7 812E5

1E5

80000

60000

40000

20000

0

MK2

MK2

DEN

V

MK2

DEN

V S

GH

MK2

DEN

V S

GH

sialid

ase

CHO

WT

CHO

WT

DEN

V

CHO

WT

DEN

V S

GH

CHO

WT

DEN

V S

GH

sialid

ase

MeanMean plusmn SD

(a)

12E5

1E5

0 5 10 20 40 80

SG

80000

60000

40000

20000

MeanMean plusmn SD

(b)



4 Discussion















Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Sialic Acid Expression in the Mosquito