, Abdul R. Omar , Chong P. Pei , Zamberi Sekawi

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, Abdul R. Omar , Chong P. Pei , Zamberi Sekawi

aMedicalLaboratoryTechnology,FacultyofAppliedMedicalScience,NorthernBorderUniversity,Arar,SaudiArabia

bDepartmentofMedicalMicrobiologyandParasitology,FacultyofBasicClinicalSciences,CollegeofHealthSciences,UsmanuDanfodiyoUniversity,Sokoto, 840232SokotoState,Nigeria

cDepartmentofPharmacyPractice,CollegeofPharmacy,AlMaarefaUniversity,Dariyah,13713,Riyadh,SaudiArabia

dDepartmentofMedicalLaboratorySciences,SulaimanALrajhiUniversity,Albukairah,SaudiArabia

eSchoolofBiosciences,Taylor’sUniversity,No1,JalanTaylor’s,47500SubangJaya,Selangor,Malaysia

fDepartmentofVeterinaryPathologyandMicrobiology,FacultyofVeterinaryMedicine,UniversityPutraMalaysia,43400Serdang,Selangor,Malaysia

gDepartmentofMedicalMicrobiologyandParasitology,FacultyofMedicineandHealthSciences,UniversitiPutraMalaysia,43400Serdang,Selangor, Malaysia

a rt i c l e i nf o

Articlehistory:

Received11July2021

Receivedinrevisedform28August2021 Accepted1September2021

Keywords:

Antibodies Bioinformatics Cross-protection Reversevaccinology Rhinoviruses Vaccine

a b s t ra c t

Background:Rhinoviruses(RV)areassociatedwiththedevelopmentandexacerbationsofasthmaand chronicobstructivepulmonarydisease.They’vealsobeenlinkedtomoreseverediseaseslikepneumonia, acutebronchiolitis,croup,andotitismedia.Becauseofthehypervariablesequencesinthesameserotypes, noeffectivevaccineagainstrhinoviruseshasbeendevelopedtodate.Withtheavailabilityofnewfull- lengthgenomesequencesforallRV-AandRV-Bserotypedstrains,thisstudyusedbioinformaticstoﬁnd asuitableRVstrainwiththehighestsimilaritymatricestotheotherstrains.

Methods:ThefullgenomicsequencesofallknowndifferentRV-Aand-Bprototypesweredownloaded fromtheNationalCentreforBiotechnologyInformation(NCBI)anddividedintominorlow-density lipoproteinreceptor(LDLR)andmajorintercellularadhesionmoleculegroups(ICAM).Thesequences wereeditedusingBiologicalSequenceAlignmentEditor,v7.2.0(BioEditsoftware)tostudyeachcapsid protein(VP1,VP2,VP3,andVP4)andanalyzedusingtheEMBL-EBIClustalWserverandthemorecurrent ClustalOmegatoolforthecalculationoftheidentitiesandsimilarities.

Results:Weanalyzedandpredictedimmunogenicmotifsfromcapsidproteinsthatareconservedacross distinctRVserotypesusingabioinformaticstechnique.TheaminoacidsequencesofVP3werefound tobethemostvaried,whileVP4wasthemostconservedproteinamongallRV-AandRV-Bstrains.

Amongallstrainsstudied,RV-74demonstratedthehighestdegreeofhomologytootherstrainsand couldbeapotentialgeneticsourceforrecombinantproteinproduction.Ninehighlyconservedregions withaminimumlengthof9-merswereidentiﬁed,whichcouldserveaspotentialimmunetargetsagainst rhinoviruses.

Conclusion:Therefore,bioinformaticsanalysisconductedinthecurrentstudyhaspavedthewayfor theselectionofimmunogenictargets.Bioinformatically,theidealstrain’scapsidproteinissuggestedto containthemostcommonRVsimmunogenicsites.

org/licenses/by-nc-nd/4.0/).

∗Correspondingauthorat:FacultyofScience,NorthernBorderUniversity,Saudi Arabia.

E-mailaddresses:[email protected]

(A.S.Alshrari),[email protected](S.A.Hudu),[email protected](S.M.B.Asdaq), [email protected](A.M.Ali),[email protected](C.V.Kin),[email protected] (A.R.Omar),[email protected](C.P.Pei),[email protected](Z.Sekawi).

Introduction

Rhinoviruses(RVs),whichwereformerlyknownashumanrhi- noviruses,wereﬁrstisolatedin 1956[1]. RVs representalarge number of small non-enveloped viruses of about 28–30nm in diameterwithinthegenusEnterovirusofthePicornaviridaefam- ily[2].Theviralgenomeisapositive-sensesingle-strandedRNA (+ssRNA)ofapproximately7200bases[3].Within50yearssince

https://doi.org/10.1016/j.jiph.2021.09.001

1876-0341/©2021TheAuthor(s).PublishedbyElsevierLtdonbehalfofKingSaudBinAbdulazizUniversityforHealthSciences.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Fig.1. SimilarityofHRV-74VP1aawithotherserotypeswithinRV-A.HRV-74VP1identitywithintheRV-Agrouprangedfromalowof61.62%(HRV-8)toahighof92%

(HRV-15)withamedianidentityof75%.B.ThesimilarityofHRV-74VP2(aa)withotherserotypeswithinRV-A.HRV-74VP2identitywithintheRV-Agrouprangedfrom alowof72.03%(HRV-8)toahighof94.27%(HRV-15)withamedianidentityof81.37%.C.ThesimilarityofHRV-74VP3aawithotherserotypeswithinRV-A.HRV-74VP3 identitywithintheRV-Agrouprangedfromalowof67.23%(HRV-45)toahighof91.6%(HRV-15)withamedianidentityof78.24%.D.ThesimilarityofHRV-74VP4aawith otherserotypeswithinRV-A.HRV-74VP4identitywithintheRV-Agrouprangedfromalowof89.86%(HRV-71)toahighof100%(24serotypepairs)withamedianidentity of97.35%.

theirdiscovery,RVshavebeendividedintothreegroups,RV-A,-B, andC,withthelatter,RV-C,beingreportedrelativelyrecentlyin 2007[4].Withineachgrouporspecies,therearemultipleRVsvari- ablydesignatedas“serotypes,”“types,”or“strains.”Therearemore than100typesofRV-AandB,whilethediscoveryofnewRV-Ccon- tinues[4–6].Theviralcapsid,whichsurroundsthegenomicRNA, iscomposedof60identicalcopieseachoffourstructuralproteins, designatedasVP1,VP2,VP3,andVP4.Thethreemoreabundant proteins(VP1,VP2,andVP3)areexposedonthecapsidsurfaceand accountforthevirus’antigenicdiversity,whilethesmallestone (VP4)liesattheinterfacebetweenthecapsidandtheviralgenome.

Amongthefourcapsidproteins,VP1isthelargestandthemost exposed,anditservesasthesiteofattachmenttothecellsurface receptors[7,8].ThesurfaceoftheRVscapsidcontainsneutraliza- tionantigenicandhostcell-bindingsites,whichallowthevirusto startitsreplicationcyclebyattachingtothehostcellreceptors[9].

Many molecularepidemiologicalstudiesofRVsconductedin different regions have revealed that there are no predominant circulatingserotypesthatcouldbeconsideredforvaccinedevelop- ment[10,11].WiththehighRVburden,whichispoorlyresponsive to the current therapies, alternative approaches to overcome theirinfectionsarethereforeneeded.Elicitingcross-neutralizing antibodies has been considered the ¨holy grail¨in thesearch for effectiveRVvaccines.Capsidproteins(VP1−4)orantigenicpep- tidescorrespondingtooneofthemhavebeenclaimedtoinduce cross-neutralizingantibodiesagainstdifferentRVstrains[12,13].

Hence,alternativestrategieswereappliedinanattempttodesign abroad-spectrumRVvaccinebasedontheoppositeapproach.In theeraofgenomics,thestartingpointofdesigninganidealvac- cineagainstRVscouldbefromtheavailableinformationontheir genomes. Recently,thefull-lengthgenomesequencesofallRV- AandRV-Bserotypedstrainshavebeenreported[14].Therefore,

thisstudyaimedtousebioinformaticsapproachestocharacterize thesequencesofthefourcapsidproteins(VP1,VP2,VP3,andVP4) ofknownRV-AandRV-Bserotypestodeterminethephylogenetic relationshipbetweenstrainsandtorevealtheidealstrainthatpos- sessesgenomesequencesthatarehighlyidenticaltootherstrains andultimatelytoidentifythehighlyconservedantigenicregions acrossthemajorityofRVsAandBwithinthecapsidproteinsto serveasvaccinecandidatesinfuturestudies.

Materialsandmethods Datacollection

The complete genome sequences of all known distinct RV- A and-B prototypes downloaded from the National Centre for BiotechnologyInformation(NCBI)(http://www.ncbi.nlm.nih.gov) exceptHRV-87asithadbeenreclassiﬁedasahumanenterovirus [15]. VP1 data under GenBank accession number AY355180 to AY355281,asdescribed[16],wasadaptedtoconﬁrmtheaccuracy oftheanalysis.

Characterizationofrhinovirus(RV)capsidproteinVP1,VP2,VP3, andVP4

TheobtainedRVsequences(RV-A,n=75;RV-B, n=25)were grouped based on theiroriginal classiﬁcation. The RV-A group wasdividedintotwosub-groups:minorlow-densitylipoprotein receptor(LDLR)(n=12)andmajorintercellularadhesionmolecule (ICAM)(n=63).Upongrouping,thesequenceswereeditedusing BiologicalSequenceAlignmentEditor,v7.2.0(BioEditsoftware)to studyeachcapsidprotein(VP1,VP2,VP3,andVP4)independently [17].Sequence alignmentswere performedfor alltheavailable

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Miningforconservedsequencesamongthealignedsequences wasperformedbydeterminingtheentropyofregionswithatleast nineaminoacidsinthelengthandmaximumaverageentropyof 0.2.Themaximumentropyperpositionwas0.2,withgapslimited to2persegment.Contiguousgapswerelimitedto1inanyseg- ment.Thisminimumlengthofaminoacidswaschosenbecauseit representsthebindingcorelengthofamajorityofhumanleukocyte antigen(HLA)restrictedT-celldeterminants[19,20].

B-cellepitopeprediction

Thereareseveralbioinformaticstoolsforassessingandpredict- ingBcellepitopes[14,21].Wechosetodeterminethecontinuous Bcellepitopesasitismainlybasedonthepropertiesoftheamino acidsinthepeptide,suchasthehydrophilicity,charge,exposed surfacearea,andsecondarystructure.Fordiscontinuousepitope prediction,apre-requisiteisthe3-Dstructureoftheantigen.We usedtheABCPredsoftware(www.imtech.res.in/raghava/abcpred/

)forcontinuousB-cellepitopeprediction.Brieﬂy,theaminoacid sequenceoftheVPcandidategeneswasuploadedtotheABCPred servertobetested againsta preselecteddatasetof 700unique experimentally proven B-cellepitopes,which is asubset ofthe BCIPEPdatabase,aswellas700non-epitopesorrandompeptides.

Thedefaultthresholdwaschosenfortheanalysis.

Results

PhylogeneticanalysisoftheretrievedsequencesoftheRVcap- sid proteinswas performedfor the future goal of constructing recombinant proteinsfor useas vaccinecandidates. Thisfacili- tatedthedeterminationoftherelationshipsbetweenstrainsand identiﬁcationofthe ¨ideal¨strainamongstrainsthatarehighlyiden- ticaltootherstrains.Also,thepriorknowledgeofthevariability among prototype strainshasprovided a unique opportunityto enhancetheoptimalselectionoftheimmunogens,andafterward, theselectionofstrainsexaminedforcross-reactivity.Alignment analysisshowedthatfull-lengthVP1,VP2,andVP3aminoacid(aa) sequenceswerefoundtobevariablebetweenstrainsandvaried inlength,withVP4astheexception.VP4possessedtheshortest sequencewithonly69aaresiduesinlengthandwasfoundtobe themostconservedproteinamongallcapsidproteins.

CharacterizationofVP1sequence

Thepair-wiseanalysisrevealedthatVP1aminoacidsequences withinindividualgroupswerenotwellconservedwithanaverage identityof70.61%forRV-A,whichrangedfrom57.24%(HRV-45and HRV-49)to98.59%(HRV-8and-95).Ontheotherhand,theaverage identityofVP1aawithinRV-Bwas76.27%,witharangeof66.9%

(HRV-14and-84)to93.24%(HRV-70and-91).Therewereeighteen

againsttheconsensusVP1sequence.

Fig.2 depicts thesequence alignment of theHRV74VP1 aa sequenceagainsttheconsensusVP1sequence.

CharacterizationofVP2sequences

ThelevelofVP2 aaidentitywithinRV-Arangedfrom70.11%

(HRV-23and-8)to99.23%(HRV-8and95)withamedianidentityof 80.27%.Meanwhile,RV-BVP2aminoacidsequenceidentityranged from73.95%(HRV-79and84)to96.18%(HRV-70andHRV-17)with amedianidentityof81.28%.Therewereeightyandtwenty-four serotypepairsthathaveanaminoacidsequenceidentityofmore than90%forRV-Aand-B,respectively.Also,aconsensussequence (270aa)thatrepresentedthe75alignedVP2 aasequenceswas obtained.TheconsensusidentitywithintheRV-Agroupranged from74.7%(HRV-8)to91.92%(HRV-21),withamedianidentityof 84.7%.Ouranalysisalsoshowedthat130residues(48.14%ofthe VP2fulllength)atdifferentpositionswere100%conservedacross allRV-A.Fig.3showsthesequencealignmentofHRV74VP2aato theconsensussequence.

WithinRV-A,thelevelofVP3aaidentityrangedfrom63.14%

(HRV-30and8)to100%(HRV-8and95)withamedianidentityof 76.11%.Ontheotherhand,theRV-Baminoacidsequenceidentity oftherespectiveproteinrangedfrom71.61%(HRV-3and27)to 97.88%(HRV-70andHRV-17)withthemedianidentityof81.38%.

Thereweresixty-one,andtwenty-sevenserotypepairsthathave anaminoacidsequenceidentityofmorethan90%forRV-Aand -B,respectively.Besides,thecconsensussequence(241aa) that representedthe75alignedVP3aasequencesofRV-Aserotypeswas generated.TheconsensusidentitywithintheRV-Agroupranged from72.27%(HRV-8and95)to88.24%(HRV-43)withamedian identityof80.38%.Ouranalysisalsoshowedthat90residues(37.3%

oftheVP3fulllength)atdifferentpositionsare100%conserved acrossallRV-A.ThesequencealignmentofHRV74VP3aatothat oftheconsensussequenceisshowninFig.4.

Lastly,withinRV-A,thelevelofVP4aaidentityrangedfrom 88.41%(severalserotypes)to100%(332serotypespairs)witha medianidentityof95.91%.Meanwhile,RV-Baminoacidsequence identityrangedfrom88.41%(HRV-26and48)to100%(5serotype pairs)withamedianidentityof95.3%,otedthatVP4istheonlycap- sidproteinwhosemedianidentityofserotypepairswithin-group aismorethangroupB.Incontrasttoothercapsidproteins,only seventy-sixandsevenserotypepairshaveanaminoacidsequence identitylessthan90%forRV-AandB, respectively.Besides,the

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Fig.2.AlignmentoftheRV-AVP1consensussequenceandHRV-74VP1sequence.Theconsensussequencewasbuiltbasedonthe75full-lengthRV-AVP1aasequences.An

*(asterisk)indicatespositionsthathaveasingle,fullyconservedresidue.A:(colon)indicatesconservationbetweengroupsofstronglysimilarproperties.A.(dot)indicates conservationbetweengroupsofweaklysimilarproperties.

Fig.3. AlignmentofconsensusoftheRV-AVP2sequenceandHRV-74VP2sequence.Thealignmentshowsahomologyof87.2%betweenbothsequences.Theconsensus wasbuiltbasedonthe75full-lengthRV-AVP2aasequences.An*(asterisk)indicatespositionsthathaveasingle,fullyconservedresidue.A:(colon)indicatesconservation betweengroupsofstronglysimilarproperties.A.(dot)indicatesconservationbetweengroupsofweaklysimilarproperties.

Fig.4.AlignmentofconsensusoftheRV-AVP3sequenceandHRV-74VP3sequence.Thealignmentshowshomologyof87%betweenbothsequences.Theconsensuswas builtbasedonthe75full-lengthRV-AVP3aasequences.An*(asterisk)indicatespositionsthathaveasingle,fullyconservedresidue.A:(colon)indicatesconservation betweengroupsofstronglysimilarproperties.A.(dot)indicatesconservationbetweengroupsofweaklysimilarproperties.

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aasequenceofVP4.Furtheranalysisalsoshowedthat55residues (79.7%oftheVP4fulllength)atdifferentpositionsare100%con- servedacrossallRV-A.

IdentiﬁcationofpotentialrhinovirusVP1,VP2,VP3,andVP4 conservedregions

Inthisstudy,uponmultiplealignmentsofVP1aasequences, severalconservedregionsacrosseachselectedgroupwithamini- mumlengthof9residuesandmaximumentropy0.2werefound.

Ouranalysisshowedthattherewerethreeconservedregionsinthe RV-Amajorreceptorgroup,whilesixmotifsweredetectedamong theminorreceptorgroup(Table1).Uponmultiplesequencealign- mentofallRV-A,weidentiﬁedthreehighlyconservedregionsfor theVP1I-CAM1 region(UnderlinedinTable1).Meanwhile,our analysisofRV-Bshowednoconservativemotifsusingtheapplied criteriaasdescribed.However,severalconservedmotifswithinthe groupwere observedoncethemaximumentropy waschanged to0.5,asshowninTable1.Also,severalresiduesappearedtobe conservedacrossallstudiedRVprototypestrains(RV-A&-B).

OuranalysisoftheVP2aasequenceindicatedthattheVP2aa sequencewasmoreconservedandshowedlessvariabilityinboth groupsascomparedwithVP1protein.Severalconservedregions acrosseachselectedgroupwithaminimumlengthof9residues andmaximumentropyof0.2werefound.TheanalysisoftheRV-A majorreceptorgroupshowedthreeconservedregions,whileseven motifsweredetectedamongtheminorreceptorgroup(Table2).

Unlike VP1, VP2 of RV-Bshows sixconserved motifs usingthe appliedcriteria,asshowninTable2.Also,severalresidueswithin thedetectedregionsappeartobeconservedacrossallstudiedRV (RV-A&-B)prototypestrains(Table2).

OurfindingsrevealedthattheVP3aasequenceswerethemost variableamongtheVP1–4capsidproteins.Unlikeothercapsidpro- teins,noconservedregioningroupAwithaminimumlengthof9 residuesandmaximumentropy0.2wasfound.However,oncewe modifiedtheselectionto9residuesandmaximumaverageentropy of0.2withdisablingofthemaximumentropyperposition,five notsowell-conservedmotifswereobservedwithinRV-AICAM-1 group(Table3).Ontheotherhand,theanalysisoftheminorrecep- tor(LDLR)groupproteinshowsfivehighlyconservedregions,with onlytwodetectedamongRV-Bfortherespectiveprotein(Table3).

Besides,therewerefewerresidueswithinthedetectedregionsthat appeartobeconservedacrossallstudiedRV(RV-A&-B)prototype strains(highlightedinTable3).

In contrast with VP3, our results showed that the VP4 aa sequencewasthemostconservedcomparedtotheotherfourVPs.

Ouranalysisshowedthat72%oftheVP4sequenceishighlycon- servedwithintheHRV-Aprimaryreceptor.Furthermore,VP4 of theminorreceptor(n=10)containedthreehighlyconservedseg-

againstRVs.

ThepredictedBcellepitopesarerankedaccordingtotheirscore obtainedbythetrainedrecurrentneuralnetwork.Ahigherscoreof thepeptidemeansahigherprobabilityofbeingasanepitope.All thepeptidesshownwith*areabovethethresholdvaluechosen.

Discussion

Inthisstudy,weadoptedbioinformaticsanalysisonavailable RVprototypestrainsintendingtocharacterizeandunderstandthe relationshipbetween strains, toidentifya potentstrain that is highlyandideallysimilartootherstrains,andtoanalyzeallthe immunogeniccapsidproteinsofallstrainstoidentifythehighly conservedaminoacidsequences.Throughaasequenceanalysis,the fourcapsidproteins(VP1−4)werefoundtobevariableinlength andsequenceamong differentstrains. Ourresultsrevealedthat VP3isthemostvariableprotein,followedbyVP1andVP2pro- teins,whileVP4isthemostconservedprotein.Ourﬁndingsinthis studyareinagreementwithpreviousstudies[23,24].

Ourfindingsdemonstrated thatwithinthegroup,theamino acidsequencesofVP4havethemostsignificantdegreeofidentity, rangingfromaslowas88.41%(severalserotypepairs)toashigh as100%(337serotypepairs).EventhoughVP4islocatedinside thevirion,thebioinformaticsfindingsstronglysuggestthatVP4 maybeconsideredanimmunogenicparticle.Apreviousstudy[26]

showedthatantibodiesagainsttheN-terminusofVP4HRV14are capableofneutralizingviralinfectivity.Anotherstudy[25],also demonstratedthattheHRV14capsidisdynamicandtransiently displaystheburiedN-terminiofviralVP1andVP4bybreathing phenomenon.Meanwhile,arecentstudyalsoindicatedthatVP0 (VP4+VP2)inducescross-reactiveimmuneresponses.Hence,itis stronglysuggestedthatVP4couldbeacandidatefortheRVvaccine [27].Meanwhile,VP1−3proteinsareknowntobelocatedonthe surfaceoftheRVcapsids[28].Hence,theseVP1−3capsidproteins areexpectedtobeamenabletovaccinedevelopment.However,our ﬁndingsrevealedthattheyarehighlyvariableantigenicproteins, whichcouldhindertheirpotentialasvaccinecandidatesthatcould elicitcross-neutralizingantibodiesinourcase.Furthermore,there islimitedevidencefortheirimplicationsinvaccination.Previous studiesreportedthatVP1proteinhadbeenconsideredashaving thehighestpotentialasavaccinecandidate[8,29]becauseitisthe mostsurface-exposedandimmunodominantofallthefourcapsid proteins.

Thecryoelectronmicroscopyofrhinovirus14virionscontains theoctanucleotidehigh-densitysegmentoftheRNAgenomethat bindswiththeVP-2subunits.Ithasbeenshownthatthebindingof ICAM-1torhinovirus14isneededtopreparethevirusforgenome dischargeatacidicpH.Arrangementoftherhinovirus14–ICAM-1 complexincitesconformationalchangestotherhinovirus14cap-

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Table1

ConservedsequencesidentiﬁedwithinRV-VP1capsidproteins.

Sp. Receptor No. Peptidelength Region** Consensus

RV-A

ICAM-1

1 9 11to19 LNEVLVVPN

2 9 34to42 LDAAETGHT

3 11 197to207 ASAYYMFYDGY

LDLR

1 11 9to20 EVLNEVLVVPNI

2 16 34to49 LDAAETGHTSNVQPED

3 9 68to76 ESFLGRSGC

4 10 103to112 LQEMAQIRRK

5 11 143to153 MQYMYVPPGAP

6 11 190to200 ASAYYMFYDGY

RV-B ICAM-1

1 9 70to78 FLGRAACVH

****:****

2 10 114to123 LSSLVQLRKK

:*****:*:*

3 15 127to141 FTYVRFDSEYTILAT

***.********:**

4 14 173to186 DYTWQSASNPSVFF

*:*****:******

5 14 202to215 ASAYNCFYDGYSHD

****..********

6 10 221to230 YGINVLNHMG

***:****** Sp.:Species.No.:PeptideNo.detectedineachsub-group.(**):Numberingofaminoacidsisbasedonconsensussequencegeneratedforthestudiedprotein.Theunderlined sequenceistheconsensusofhighlyconservedmotifsacrossallRV-A(n=75).HighlightedresiduesareconservedacrossallstudiedRV-A&-B(n=100).An*(asterisk)indicates positionsthathaveasingle,fullyconservedresidue.A:(colon)indicatesconservationbetweengroupsofstronglysimilarproperties.A.(dot)indicatesconservationbetween groupsofweaklysimilarproperties.

Table2

ConservedsequencesidentiﬁedwithinRV-VP2capsidproteins.

Species Receptor No. Segmentlength Region** Consensus

RV-A

VP2-ICAM

1 9 4to12 VEACGYSDR

2 10 77to86 GWWWKLPDAL

3 11 207to217 PYVNAVPMDSM

VP2-LDLR

1 10 4to13 VEACGYSDRI

2 17 15to31 QITRGDSTITSQDVANA

3 11 56to66 PDTSSNRFYTL

4 12 75to86 SKGWWWKLPDAL

5 11 173to183 WLNFDGTLLGN

6 14 188to201 PHQFINLRSNNSAT

7 11 205to215 PYVNAVPMDSM

RV-B VP2-ICAM

1 9 5to13 EACGYSDRV

2 12 19to30 GNSTITTQEAAN

3 13 75to87 SKGWCWKLPDALK

4 12 109to120 HVQCNATKFHSG

5 16 183to198 IFPHQFINLRTNNTAT

6 13 202to214 PYINSVPMDSMTRHNN

**:**.*:******** (**):Numberingofaminoacidsisbasedontheconsensussequencegeneratedforthestudiedprotein.Theunderlinedsequenceistheconsensusoffullyconservedmotifs acrossallRV A(n=75).HighlightedresiduesareconservedacrossallstudiedRV A&-B(n=100).An*(asterisk)indicatespositionsthathaveasingle,fullyconserved residue.A:(colon)indicatesconservationbetweengroupsofstronglysimilarproperties.A.(dot)indicatesconservationbetweengroupsofweaklysimilarproperties.

sid,includingthemovementoftheCendsofVP4subunits,which becomereadyfordischargethroughporesthatopeninthecap- sidsofinitiatedparticles.[30].However,furtherdetailsforVP1and othersremainelusiveeventhoughseveralstudieshavebeencon- ductedfordecades[31].VP2ishighlysuggestedtobeconsidered inthevaccinationapproach. Inparticular,VP2 hasbeenconsid- eredin severalstudiesasa targetfor RVvaccinedevelopment, anti-peptidesderivedfromVP2showedneutralizingactivityand havebeensuggestedtoplayaroleinthefutureofRVvaccination [32–34].VP3isquitedifferentfromVP2.First,itisshorter(236–241 aa)butismorevariableincontrasttoVP4,whichisshorter(69 aa)butmoreconserved.ThemedianidentityofVP3withingroup Ais76.1%,whileitis80.3%forB.Theanalysiscarriedoutshows thatthereare90residues(37.3%ofthefulllength)thatarefully conservedacrossallRV-A.Intermsofnumbers,VP3ismorecon- servedthanVP1,butnowell-conservedmotifswithnineresidues ormorewereidentiﬁed.Thisfeaturehasalloweditscategorization (VP3)asthemostvariableproteinamongthefourstudiedpro-

teins.Justlikethebioinformaticsanalysis,limitedstudiesexplain theroleofVP3inimmunogenicity.Veryrecently,thosewhoinves- tigatedthespeciﬁcitiesofantibodyresponsesinindividuals(with andwithoutasthmaafterexperimentalHRV-16infection)foundno relevantantibodyresponsesspeciﬁcforeitherVP3orVP4.Another studyinvestigatedthemosthighly immunogenicneutralization antigensusingneutralizingmonoclonalantibodiesraisedagainst nativevirion[35,36].Thisisoneoftheearlypiecesofevidence thatshowsVP1-VP3playingaroleinRVsimmunogenicity.Todate, NimIA-NimIIIhasbeendisplayedasraisedfeaturesinallresolved structuresofRV-Aand-Bstrains. IncontrasttoVP1,thephysi- calloopsofHRV-C15forputativeNimIIandNimIIIanalogswere foundtobesimilartoHRV-A16andHRV-B14[37].Incontrasttothe threesurface-exposedproteins,onlyseventy-six(2.7%)andseven (2.3%)serotypepairshadanaminoacidsequenceidentityofless than90%for VP4of RV-AandB, respectively.Interestingly,the averageofmedianidentitiesintherespectivegroupswas95.91%

(90.95%–97.35%)versus95.3%.Itistheonlycapsidproteinwhose

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B VP3-ICAM 2 12 116to127 MYTGPALSSAKL (**):Numberingofaminoacidsisbasedontheconsensussequencegeneratedforthestudiedprotein.NofullyconservedmotifsacrossallRV-A(n=75).Highlightedresidues areconservedacrossallstudiedRV-A&-Bserotypes(n=100).An*(asterisk)indicatespositionsthathaveasingle,fullyconservedresidue.A:(colon)indicatesconservation betweengroupsofstronglysimilarproperties.A.(dot)indicatesconservationbetweengroupsofweaklysimilarproperties.

Table4

ConservedmotifsidentiﬁedwithinRV-VP4capsidprotein.

SpeciesGroupReceptor No. Segmentlength Region** Consensus

RV-A

VP4-ICAM

1 17 1–17 MGAQVSRQNVGTHSTQN

2 17 22–38 GSSLNYFNINYFKDAAS

3 16 46–61 FSQDPSKFTDPVKDVL

VP4-LDLR

1 17 1–17 MGAQVSRQNVGTHSTQN

2 20 19–38 VSNGSSLNYFNINYFKDAAS

3 22 48–69 QDPSKFTDPVKDVLEKGIPTLQ

RV-B VP4-ICAM 1 12 27–38 FTVINYYKDAAS

2 13 47–59 SMDPSKFTEPVKD

(**):Numberingofaminoacidsisbasedontheconsensussequencegeneratedforthestudiedprotein.HighlightedresiduesareconservedacrossallstudiedRV-A&-B serotypes(n=100).

Table5

PredictionofB-cellepitopeswithinconservedregionsidentiﬁedwithinVP1,VP2,VP3,andVP4proteinsequencesfromABCPredPredictionSoftwareServer.

[22]Rank Sequence Startposition Score

VP41* MGAQVSRQNVGTHSTQN 2 0.64

MGAQVSRQNVGTHSTON 1 0.61

MGAQVSRQNVGTHSTQN 5 0.57

VP42* GSSLNYFNINYFKDAAS 1 0.78

GSSLNYFNINYFKDAAS 2 0.62

GSSLNYFNINYFKDAAS 5 0.58

GSSLNYFNINYFKDAAS – –

VP43* FSQDPSKFTDPVKDVL 2 0.77

FSQDPSKFTDPVKDVL 3 0.64

VP21* GWWWKLPDAL 1 0.67

VP22* PYVNAVPMDSM 2 0.55

VP11 ASAYYMFYDGY – –

medianidentityofserotypepairswithingroupAwasmorethan thatofgroupB.Withinthegroup,HRV-74VP4showedamedian identityof97.35%with24serotypesthatare100%identical.Inter- estingly,alltheRV-Aserotypesexceptforone(A71)showidentity by90%ormoretoHRV-74VP4.

Interestingly, ourcurrent results demonstrated that HRV-74 hasthehighestidentityandhomologyascomparedtootherHRV strainsamongallcapsidproteinsstudied.Thisisanexcellentindi- catortoemploysuchastraininfutureRVvaccinedevelopment.

Forexample,HRV-16and-89havebeenselectedasthestrainsof choiceforvaccineprojectsinpreviousstudies[38].Intheirstudies, within-groupA,themedianidentityofHRV-16andHRV-89is70%

and 67.7%,respectively.However,inourstudy,HRV-74showed morethan80%identityinallcapsidproteinsstudied.Hence,itis highlysuggestedthatHRV-74couldbeabetterchoiceforthedevel-

opmentofvaccines.Signiﬁcantsequencevariationsbetweenthe fourcapsidproteinsoftheRVserotypeshavealwaysbeenanissue inthedevelopmentofavaccinethatprovidespotentprotection againsteachserotype.Therefore,ahighlyconservedregionacross apathogenstrainhasalwaysbeenconsideredtobeanexcellent choicetosolvesuchanissue[39,40].

The N-terminus of VP1 and VP4 in several closely related Picornaviruseshasbeensuggested tobeexternalizedduringthe uncoatingprocess.Together,theyallowtheviralparticletointeract directlywiththehostcellbyshapingaporeinthecellmembrane, throughwhich theviralRNAis releasedtothecytoplasm [41].

Ifthisisthecase,blockingsucha viralsite byanantibodywill probablypreventtheviralgenomefrombeingtranslocatedtothe hostcytoplasmand,consequently,viralinfectivity.Apartfromthat, andmoreinterestingly,mostresiduesofthreeconservedmotifs

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detectedinRV-AVP2arealsohighlyconservedinRV-B.Justlike VP1,twooftheidentiﬁedmotifsarelocatedattheVP2N-terminus, while thethird (207PYVNAVPMDSM217)isat theC-terminus.

Unlikebothproteins,theVP4aasequencewasfoundtobethemost conservedprotein.

Interestingly,theanalysisshowsthat72%and85%oftheVP4 sequence is highly conserved amongthe HRV-Amajorand the minorreceptorgroups,respectively.HRV-BVP4,incontrast,con- tainslessconservedregions,whichexhibitonly25%oftheprotein’s total length. Upon multiple sequence alignment of all HRV-A, three highly conserved regions were identified forVP4. The B- cell epitopespredictedfromtheABCPred servercouldserve as potentialexperimentalvalidationtargetsfortheRVvaccinedevel- opment.Therefore,ourfutureworkwillbeaninvivostudythat involvesimmunogenicityandhumoralresponsesinanimalprotec- tionexperiments,serologicandfunctionalassayssoastodetermine theefficacyofthepeptidevaccineandfileapatentofthesame.

Conclusions

In conclusion,this study,therefore,identiﬁedand character- izedhighlyconservedregionswithaminimumlengthof9-mers withintheVP1,VP2,andVP4 proteinsequencesusingbioinfor- matics.TheseconservedregionsacrossthemajorityofRV-A,and mayrepresentpotentialimmunetargets.Remarkably,thesecon- servedregionshaveshownstabilityovertheentirehistoryofRV-A sequencesdepositedintheNCBIdatabase,asillustratedbytheir lowentropyvaluesandvariantfrequencies.Therecombinantpro- teinscouldbepotentialcandidatevaccineagainstRVinfectionin futurestudiesaftermoreextensiveresearchsuchasinvivoanimal modeltestingandotherprocedures.

Funding

Nofundingsources.

Competinginterests Nonedeclared.

Ethicalapproval Notrequired.

Acknowledgment

ThisworkwassupportedbytheMinistryofHigherEducation, Malaysia,throughtheFundamentalResearchGrantScheme(FRGS) (Grantnumber-5524211).

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