Detection and genetic characterization of Mamastrovirus 5 from Brazilian dogs

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Detection

and

genetic

characterization

of

Mamastrovirus

5

from

Brazilian

dogs

Christian

D.B.T.

Alves

_,

_Renata

_F.

_Budaszewski

_,

_Marcela

_Torikachvili

_,

André

F.

Streck

_,

_Matheus

_N.

_Weber

_,

_Samuel

_P.

_Cibulski

_,

_Ana

_P.

_Ravazzolo

_,

Vagner

R.

Lunge

_,

_Cláudio

_W.

_Canal

a_{UniversidadeFederaldoRioGrandedoSul(UFRGS),FaculdadedeVeterinária,LaboratóriodeVirologia,PortoAlegre,RS,Brazil} b_{UniversidadedeCaxiasdoSul(UCS),FaculdadedeMedicinaVeterinária,LaboratóriodeImunologia,CaxiasdoSul,RS,Brazil} c_{UniversidadeFederaldoRioGrandedoSul(UFRGS),FaculdadedeVeterinária,LaboratóriodeImunologiaeBiologiaMolecular,Porto}

Alegre,RS,Brazil

d_{UniversidadeLuteranadoBrasil,PróReitoriadePesquisaePósGraduac¸ão,LaboratóriodeDiagnósticoMolecular,Canoas,RS,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Received16March2017 Accepted26September2017 Availableonline2February2018 AssociateEditor:MauricioNogueira

Keywords: Mamastrovirus5 Canineastrovirus Dog Gastroenteritis MAstV5

a

b

s

t

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Mamastrovirus 5(MAstV5),belongingtotheAstroviridae (AstV)family,previouslyknown ascanineastrovirusorastrovirus-likeparticles,hasbeenreportedinseveralcountriesto beassociatedwithviralentericdiseaseindogssincethe1980s.Astroviruseshavebeen detected in fecalsamples froma widevarietyofmammals andbirds thatare associ-atedwithgastroenteritisandextraentericmanifestations.Inthepresentstudy,RT-PCR wasusedtoinvestigatethepresenceofMAstV5in269dogfecalsamples.MAstV5was detectedin26%(71/269)ofthesamples.Interestingly,allMAstV5-positivesamplesderived fromdogsdisplayingclinicalsignssuggestiveofgastroenteritis,otherentericviruseswere simultaneouslydetected(canineparvovirus,caninedistempervirus,caninecoronavirus, canineadenovirusandcaninerotavirus).BasedongenomicsequenceanalysisofMAstV5 anovelclassificationofthespeciesintofourgenotypes,MAstV5a-MAstV5d,isproposed. PhylogeneticanalysesbasedontheORF2aminoacidsequences,samplesdescribedherein groupedintotheputativegenotype‘a’closedrelatedwithChinesesamples.Otherstudies arerequiredtoattempttheclinicalandantigenicimplicationsoftheseastrovirusgenotypes indogs.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:[email protected](C.W.Canal).

https://doi.org/10.1016/j.bjm.2017.09.008

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

VirusesbelongingtotheAstroviridae(AstV)familyare spher-ical, non-enveloped, 28–30nm in size, with a surface that formsacharacteristicstar-likestructure.1_TheRNAgenome

ofAstVrangesfrom6.8to7.9-kbinsize,polyadenylatedat the 3 end, and contains three ORFs designated as ORF1a, ORF1bandORF2.ORF1aencodesaprotease,ORF1bencodesan RNA-dependentRNA-polymerase,2,3 _{whileORF2encodesthe}

viralcapsidstructuralpolyproteinthatisrequiredforvirion assembly.4

Theviralclassificationwaspreviouslybasedonthehost andconsistedoftwogenera,AvastrovirusandMamastrovirus.

However, recent characterization ofnovel astroviruses has taken in consideration that isolates from different animal speciescanbegeneticallysimilar,whilegeneticallydiverse virusescanbeisolatedfromthesameanimalspecies.2_Based

onthisanalysis,theInternationalCommitteeonTaxonomy of Viruses renamed canine astrovirus as Mamastrovirus 5

(MAstV5).5

Astroviruses have been detected in fecal samples from a wide varietyof mammals and birds that are associated withgastroenteritis.2_{Inchildren,AstVsarethesecondmost}

commoncauseofgastroenteritisafterrotaviruses.2,6Human AstVscanalsocausesignificantdiseaseintheelderly7_and

immune-compromisedpatients.8,9_{Inadditiontoenteric}

man-ifestations,AstVshave beenassociatedwithfatalhepatitis inducks,10 _{interstitialnephritisinyoungchickens,}11

stunt-ingandprehatchingmortalityinduckandgooseembryos,12

aswellasshakingminksyndrome13_{.Recently,anAstVwas}

alsohypothesizedtobethecausativeagentofnonsuppurative encephalitisincattle.14

Since the 1980s, astrovirus-like particles have been reported in dogs with and without diarrhea.15–17 _{To date,}

canineastrovirusesorastrovirus-likeparticlesinfectingdogs have been reported in several countries.15–25 _{Despite the}

detection of MAstV5 in association with gastroenteritis in dogs,whichsuggestsapossibleroleforMAstV5asacanine entericpathogen,theassociationofMAstV5withclinical dis-easeremainsobscureinsuchreports.Here,weinvestigated thepresenceofMAstV5usingRT-PCRinfecalsamplesfrom dogsofdifferentages withand withoutdiarrhea. The par-tialgenomesofselectedMAstV5RNA-positivesampleswere alsosequencedtoperformaphylogeneticanalysiscomparing themwiththeMAstV5sequencesdescribedintheliterature asthecauseofentericdisease.18,20,23,26_{Additionally,MAstV5}

wasproposedtobeclassifiedinfourputativegenotypes.

Materials

and

methods

Samplesandnucleicacidextraction

Atotalof269dogfecalsampleswerecollectedbetween2008 and2014inveterinaryclinicsandhospitalsbyconvenience. These sampleswere obtainedfrom eight FederalStates of Brazil(Acre,MatoGrossodoSul,Paraná,RioGrandedoSul, RiodeJaneiro,Rondônia,SantaCatarinaandSãoPaulo).The animal’sagewasrecordedandrankedfrompuppy(equalor

lessthanone-year-old)toadultdog(morethanone-year-old); somesamplesfromdogsofunknownagewereincluded. Ani-malsnotpresentingdiarrheaatthetimeofsampling were consideredasymptomaticandthosepresentingclinicalsigns ofentericdisease diarrheawere classifiedassymptomatic. Samples were diluted to 20% (w/v) in phosphate buffered saline(PBS,pH7.4)andstoredat−80◦_{Cforfurtheranalysis.}

Subsequently,viralDNAisolationfromthesupernatantwas performedusingacommercialkit(NewGenePreamp®, Sim-biosBiotecnologia,Brazil)basedonguanidineisothiocyanate andsilica.27_{ViralRNAwasisolatedusingTRIzol}®_LSReagent

(Life TechnologiesTM_{,USA)according tothe manufacturer’s}

instructions.

OligonucleotidesforMAstV5detectionandsequencing

An initial screening using RT-PCR to detect a larger num-ber of Mamastrovirus species was achieved by amplifying 422bpoftheORF1bfragmentusingoligonucleotides,as pre-viously described.28 _{For the specific detection of MAstV5,}

92nucleotidesequencesofthisspecieswereretrievedfrom GenBankdatabase(http://www.ncbi.nlm.nih.gov/nucleotide), andalignedusingCLUSTALWwithinMolecularEvolutionary GeneticsAnalysisversion6(MEGA6).29TheMAstV5specific RT-PCRwasdesignedwithaprimerpairtargetingtheregionof ORF2thatamplifieda250bpfragmentselectedusingPrimer3 software.30_{Inaddition,the16SrRNAgenefromEscherichiacoli}

was amplified using the primer pair FC27 and R530as an endogenousinternalcontrolineachfecalsampleevaluated for the specific presence of MAstV5.31 _{For partial genome}

amplification, sets of12 pairs of sequencing primers were selected to amplify overlapping fragments ofORF1 (ORF1a andORF1b)andcapsidprotein(ORF2)segmentrepresentinga consensussequenceofapproximately5000nucleotides.The primersequencesareshowninTable1.

NestedRT-PCRforMAstV5detection

The cDNA was synthesized using SuperScript® III Reverse Transcriptase Kit(Life Technologies,USA)usingthe reverse primers inatotalvolumeof20␮L,followingthe manufac-turer’sinstructions.ThecDNAamplificationwasconducted inafinalvolumeof25␮Lcontaining1×PCRbuffer,1.5mMof MgCl2,0.2mMofdNTPmix,0.2␮Mofeachprimerand1unitof

Platinum®TaqDNAPolymerase(LifeTechnologies,USA).The firstroundofRT-PCRscreeningwascarriedoutwithaninitial incubationat94◦Cfor3min,30cyclesofamplification con-sistingofdenaturationat94◦Cfor1min,annealingat50◦C for1min,andextensionat72◦Cfor1min.Thesecondround wasperformedinafinalvolumeof25␮Lthatcontained2␮L ofthefirstreactionproductandthethermocyclerconditions werethesameasthoseusedforthefirstround.

The MAstV5-specific RT-PCR with specific and internal control primers was performed as a multiplex protocol. Cycling conditions were an initialcycle at94◦C for5min, 25cyclesofdenaturationat94◦Cfor30s,annealingat58◦C for 30s and polymerization at 72◦C for 1min, which was followedbyafinalextensioncycleat72◦Cfor7min.To con-firm thespecific amplificationofMAstV5, RT-PCRproducts

Table1–Oligonucleotidesequencesappliedinthepresentwork.

Primername Sequence(5–3) Target Objective Reference

ADS138F AATGTCACGGGGATACCATC ORF1a Phylogeneticanalysis Presentstudy

ADS230F GTGCATCAAACCAACACTGG ADS690R GGGTCACTCCATTCAGGAAA ADS367F CAAACCCCAACCTCAAGAGA ADS941R CATCCTCAGCAGTCCAGTCA ADS719F TGGGACACATATGGTGATGAA ADS1022R GCCTAGGCTTGAGGATGTGA ADS922F TGACTGGACTGCTGAGGATG

ADS1606R AGTCGGCTTCGGTGTCATAG ORF1b

ADS 1317F TGATCCGCTCAAATCCCTAC

ADS 1669R TTGCTCCGGACATAATCCTC

ADS1565F CTGCCTATCCCAAGATGCTC

ADS2192R GCAAATTCAAAAGCCTGGAG

ADS2173F CTCCAGGCTTTTGAATTTGC

ADS2857R ACTCTCTTGCGACCACGATT ORF2

ADS2839F ATCGTGGTCGCAAGAGAGTT ADS3535R AGTGGTTGTCCTGCTTCACC ADS3415F TTGAGCTTCACTGCACTTGG ADS4033R CATGGTGGGTTCTGTTGGTA ADS3963F CCAGCTGTTATTGGGGACAA ADS4628R TTGGTGGTGTTCTGAGGAAA ADS4446F GCCCCTGGTTCATTTTTGT ADS5030R TGAACCTGTACCCTCGATCC

ADSTTTR TTTTTTTTTTTTTTTTTTTT PoliAtail

ASTRO2 GARTTYGATTGGRCKCGKTAYGA ORF1b Screening 28

ASTRO3 GGYTTKACCCACATNCCRAA ASTRO4 CGKTAYGATGGKACKATHCC ASTRO5 AGGTAYGATGGKACKATHCC ASTRO6 GARTTYGATTGGRCKAGGTAYGA

AstVCanFor TCTGATGATGATTCTCTTCTTGATG ORF2 MAstV5specific Presentstudy

AstVCanRev GGGAACACTTTTCACGAGCA

FC27 AGAGTTTGATCCTGGCTCAG 16SRNAE.coli Internalcontrol 31

R530 CCGCGGCTGCTGGCACGTA

CPV555F CAGGAAGATATCCAGAAG VP2 CPVdetection 35

CPV555R GGTGCTAGTTGATATGTA

HA1 CGCGCTGAACATTACTACCTTGTC E3 CAdVdetection 36

HA2 CCTAGAGCACTTCGTGTCCGCTT

CCoV1F TCCAGATATGTAATGTTCGG M CCoVdetection 37

CCoV2R TCTGTTGAGTAATCACCAGCT

BEG9F GGCTTTAAAAGAGAGAATTTCCGTCTGG VP7 CRVdetection 38

END9R GGTCACATCATACAATTCTAATCTAAG

CDV1F ACTGCTCCTGATACTGC NC CDVdetection 39

CDV2R TTCAACACCRACYCCC

CDV3F ACAGRATTGCYGAGGACYTRT 40

CDV4F CARRATAACCATGTAYGGTGC

VP,virusprotein;ORF,openreadframe;E,earlyregion;M,structuralproteinM;NC,nucleocapsidprotein.

weresubmittedtopurificationusingtheNucleoSpinExtract IIKit(Macherey-Nagel,Germany)andsequenced.BothDNA strands were sequenced with an ABI PRISM 3100 Genetic Analyzerusing aBigDye Terminatorv.3.1cycle Sequencing Kit(AppliedBiosystems,USA).

Detectionofotherentericviruses

AllpositiveMAstV5sampleswerealsoscreenedforother com-monentericvirusesthroughtheamplificationofcDNA/DNA. Theprimer pairs usedforthe detectionofcanine distemper

virus (CDV),carnivoreprotoparvovirus1(canine parvovirus2, CPV2), canine coronavirus (CCoV), canine rotavirus(CRV) and,

canineadenovirus1(CAdV1)andCAdV2areshowninTable1. ThecDNA/DNAamplificationofthetargetsequenceswas con-ductedinatotalvolumeof25␮Lcontaining1×PCRbuffer, 1.5mMofMgCl2,0.2mMofdNTPmix,0.2␮Mofeachprimer

pairand1unitofTaqDNAPolymerase(LudwigBiotecnologia, Alvorada,RS,Brazil).

Genomeamplification

Four MAstV5-positive samples were selected, taking into accounttheirdifferentgeographicalorigins.ORF1a,ORF1band ORF2sequenceswereamplifiedusinganestedtouchdown RT-PCRmethod.Thefirstroundofamplificationwasconducted inafinal volumeof25␮L.Thecycling conditionsincluded aninitialdenaturationat95◦Cfor5min,20cyclesof30sfor denaturationat95◦C,outerprimerpairannealingfor30sat 55–45◦Cperthetouchdownmethod,and7minofextension at72◦C,withafinal7minextensionat72◦C.Thesamefinal volumewasusedinthesecondround,whichcontained2␮Lof theamplificationproductofthefirstround.Thesecondround cyclingconditions were aninitialdenaturation at95◦C for 5min,30cyclesof30sofdenaturationat95◦C,innerprimer pairannealingfor30sat55–45◦Cperthetouchdownmethod, and1minofextensionat72◦C,witha7minfinalextension at72◦C.

Sequencingandphylogeneticinferences

The RT-PCRproducts generated with the sets of sequenc-ing primers were purified using the NucleoSpin Extract II Kit (Macherey-Nagel, Germany). Both DNA strands were sequenced with an ABI PRISM 3100 Genetic Analyzer using a BigDye Terminator v.3.1 cycle Sequencing Kit (Applied Biosystems, USA). Overlapping fragments were aligned and assembled using SeqMan software from the DNASTAR package (DNASTAR, USA).32 _{[The open reading}

frameswere identifiedusingthe NCBIORF Findersoftware

(http://www.ncbi.nlm.nih.gov/gorf/gorf.html).]

Sequence alignment was performed using the CLUSTAL W. For the phylogenetic inferences of the MAstV capsid protein region, the MAstV5 sequences that were submit-ted togenomeamplificationand 12 MAstV5representative strainswereincluded.MEGA6software29_wasusedfor

phy-logeny inference calculated using “find best DNA/protein model” toolfrom MEGA6. TheKimura 2-parameter substi-tutionmodelwas selectedfortheMAstV5ORF2nucleotide inference, and the LG substitution model (frequencies +F) wasusedfortheaminoacidinference.Thesubstitution-rate variationamongsiteswasmodeledwithagamma distribu-tion (shapeparameter=5). Statisticalsupportwasprovided by 1000 non-parametric bootstrap analyses. A nucleotide distance matrix was calculated using an alignment with ORF2 and partialgenomesequences (Table2).The Mamas-trovirus genotypes were distinguished based on the amino acid sequence of the full length ORF2, where the genetic distances (p-dist)0.378–0.750 and 0.006–0.312 between and withingroups,respectively,wereused.5 _{Allofthesequence}

alignmentsusedtoconstructthephylogenetictreesare avail-ableinFigshare(http://figshare.com/)withtheDOI number

https://doi.org/10.6084/m9.figshare.4596325. The nucleotide

sequencesobtainedinthisstudyweredepositedinGenBank underaccessionnumbersKR349488–KR349491.

Results

DetectionofMAstV5infecalsamples

TheRT-PCRprotocolusingthescreeningprimersforMAstV5 waspositivein22%(64/269)ofthesamples,andtheprotocol usingthespecificprimersforMAstV5identified12%(32/269) ofthesamplestested. Thesamplewas consideredMAstV5 positive if the results of at least one of the two RT-PCR protocolswerepositive,whichresultedin26%(71/269)ofthe fecal samples being detected as positive. In addition, PCR

Table2–Comparisonofidentitypercentagebetweennucleotidesequenceofthepartialgenomesandaminoacid

sequenceidentitypercentageofORF2fromthesequencedMAstV5comparedwithsequencesavailableinGenBank.

Strain GenBankaccession

no.(genomesize)

Sara/13/BRA 5617/12/BRA GRAV/13/BRA 237/13/BRA

(4980nt,KR349488) (5012nt,KR349490) (5039nt,KR349491) (5011nt,KR349489) Partial genome ORF2 Partial genome ORF2 Partial genome ORF2 Partial genome ORF2 Bari/2008ITA HM045005(3120nt) 82 84 81 84 82 84 81 84 Italy/2005 FM213330(2756nt) 79 80 79 80 79 81 79 80 GI.E/Dog/ITA/2010/Zoid JN193534(2949nt) 76 77 76 77 75 77 76 77 China/2008SH8 HQ623147(2738nt) 94 97 96 97 95 96 96 97 China/2008 SH15 HQ623148(2738nt) 94 97 96 97 95 96 96 97 Gillingham/2012/UK NC026814(6617nt) 86 79 86 79 86 80 86 79 Lincoln/2012/UK KP404150(6613nt) 95 97 95 97 94 95 96 97 HUN/2012/2 KX599349(6587nt) 94 97 95 97 94 95 94 97 HUN/2012/6 KX599350(6576nt) 87 80 87 81 87 81 87 80 HUN/2012/115 KX599351(6569nt) 86 78 85 78 85 77 86 78 HUN/2012/126 KX599352(6535nt) 76 76 76 76 75 75 76 76 HUN/2012/135 KX599353(6571nt) 92 95 92 94 91 93 92 94

25 Number of MAstV5-positiv e samples 20 15 10 5 0 9 Asymptomatic Multiple infection Single infection Symptomatic 21 0 19

Fig.1–PresumptiveassociationbetweentheMAstV5-positivesamples(multipleorsingleinfection)andthepresenceof clinicalsignsinthesampleddogs.

productsgeneratedfrom bothprotocols were submitted to DNAsequencingtoconfirmtheresults(datanotshown).The RT-PCRinternalcontrolfromthe16SrRNAgeneofE.coliwas positiveinall269ofthesamplestested.

Information about clinical signs of enteric disease was availablefor49ofthe71positivesamples.Consideringonly these,inalloftheMAstV5-positivesamplesderivedfromdogs withclinicalsignssuggestiveofgastroenteritis,otherenteric virusesweresimultaneouslydetected.Detailedresultsforthe supposedassociationwithclinicalsignsandthedetectionof otherentericvirusesareshowninFig.1andTable3, respec-tively.

Animalswere rankedinagecategoryaspuppies,adults and unknown age. Adults revealed a higher frequency of

Table3–Detectionofdogentericvirusesinthe49

MAstV5-positivesamples. Virus n % MAstV5alone 21 43 CDV+MAstV5 9 18 CPV+MAstV5 3 6 CRV+MAstV5 1 2 CCoV+MAstV5 4 8 CDV+CPV+MAstV5 6 12 CPV+CCoV+MAstV5 1 2 CPV+CAV+MAstV5 1 2 CDV+CPV+CCoV+MAstV5 1 2 CPV+CCoV+CRV+MAstV5 1 2 CDV+CPV+CCoV+CAV+MAstV5 1 1 Total 49 100

MAstV5-positive RT-PCR results than puppies (puppies: 44/166,26.5%;adults:6/16,37.5%;unknownage:21/91,23.1%). NosignificantdifferencebetweendogsageandMAstV5 pos-itive samples was observed in the different ranked ages (P=0.56).

MAstV5genomesequencesandphylogeneticinferences

Four partial genomes were obtained, namely, MAstV5Sara/13/BRA, MAstV5237/13/BRA, MAstV5GRAV/13/BRAandMAstV55617/12/BRA,whichwere 4980,5011,5039and5012ntinlength,respectively,excluding the poly(A)tailandthe 5 untranslatedregions(UTRs).The threefirstpartialgenomeswerecollectedfromdifferentcities ofRioGrandedoSulState(PortoAlegre,ViamãoandGravataí, respectively),andonewasfromLondrinacity,ParanáState. Allofthefournearlycompletegenomescontainedatypical AstVorganizationinthethreepredictedORFs–ORF1a,ORF1b and ORF2. Further genome sequence comparison revealed thatthefourpartialgenomeshadgreateridentities(94–96%) with Chinesestrains, considered asthe Italy, Hungary and UnitedKingdomstrainsidentityrangedfrom76–82%,75–95% and 86–96%whencomparedtotheBrazilianstrains identi-fiedhere,respectively(Table2).Inaddition,theaminoacid sequencesoftheORF2oftheHUN/2012/126(GenBank acces-sion number KX599352), GI.E/Dog/ITA/2010/Zoid (GenBank accessionnumberJN193534)strainsshowedloweridentities (76and77%,respectively)comparedtotheBrazilianstrains

(Table2).

Phylogeneticinferenceswerealsocarriedoutwiththe par-tialandcompletesequencesofOFR2atnucleotideandamino

Z25771 L23513 AY720892 AF141381 AB013618 DQ028633 AF260508 AF056197 AB037272 Y15938 FJ890352 FM213331 NC_026814 FM213330 FM213332 KR349491 KR349488 KR349489 FJ890355 FJ222451 FJ402983 NC_013443 NC_002469 AY179509 EU847144 EU847145 EU847155 GQ502193 GQ415661 GQ415662 FJ973620 FJ571067 FJ571066 FJ571068 FJ571073 FJ571069 FJ571072 FJ571065 FJ571071 FJ571070 AB033998 FJ571074 FJ890351 KR349490 DQ344027 AY720891 DQ070852 Mamastrovirus 1 Mamastrovirus 2 Mamastrovirus 3 Mamastrovirus 4 Mamastrovirus 5 Mamastrovirus 7 Mamastrovirus 6 Mamastrovirus 8 Mamastrovirus 9 Mamastrovirus 12 Mamastrovirus 13 Mamastrovirus 10 Mamastrovirus 11 Mamastrovirus 14 Mamastrovirus 15 Mamastrovirus 16 Mamastrovirus 17 Mamastrovirus 18 Mamastrovirus 19 73 99 91 97 98 100 81 100 96 97 100 79 100 50 100 83 97 97 91 100 100 100 100 99 99 91 86 100 98 100 100 96 100 89 77 99 100 1 73

(A)

HQ623147 dogfaeces/China/2008/SH8 Mamastrovirus 5 genotype a Mamastrovirus 5 genotype b Mamastrovirus 5 genotype c Mamastrovirus 5 genotype d 0.05 99 81 96 67 98 99 68 94 99 88 92 77 HQ623148 dogfaeces/China/2008/SH15 HQ623137 dogfaeces/China/2008/SH13 HQ623145 dogfaeces/China/2008/SH21 JN985792 FRA/2012/4255-237 JN985802 FRA/2012/4255-273 KX599349 HUN/2012/2 KP404150 Lincoln/2012/UK KR349488 Sara/2013/BRA KR349489 237/2013/BRA KR349490 5617/2012/BRA KR349491 Grav/2013/BRA KC470086 BRA/2013/905 KC470088 BRA/2013/1068 KC470089 BRA/2013/1093 KC470087 BRA/2013/915 KX599353 HUN/2012/135 KX599350 HUN/2012/6 HM045005 Bari/2008 JN985790 FRA/2012/4255-91 NC 026814 Gillingham/2012/UK JN985814 FRA/2012/4255-299 JN985785 FRA/2012/4255-44 KX599351 HUN/2012/115 JN985784 FRA/2012/4255-40 JN193534 GI.E/Dog/ITA/2010/Zoid KX599352 HUN/2012/126 JN985807 FRA/2012/4255-282 GU376736 dogfaeces/China/2008/SH JQ081297 Univerified

(B)

Fig.2–EvolutionaryrelationshipofMAstV5withrepresentativeMAstVgenera.Thepercentageofreplicatesinwhichthe associatedvirusclusteredtogetherinthebootstraptest(1000replicates)isshownnexttothebranchesineachtree.The treesaredrawntoscale;barsrepresentthenumberofsubstitutionspersite.Allpositionsexceptambiguouspositionswere included.Bootstrapvalues<50wereexcluded.GenBankaccessionnumbersareshownonthetree.MAstV5sequences obtainedinthepresentstudyareindicatedwithablackdot(䊉).TheKimura2-parametersubstitutionmodelwasselected fortheMAstV5ORF2nucleotideinference,andtheLGsubstitutionmodel(frequencies+F)wasusedfortheaminoacid inference.Thesubstitution-ratevariationamongsiteswasmodeledwithagammadistribution(shapeparameter=5).(A) EvolutionarytreebasedonthecompleteaminoacidsequencesoftheORF2gene(capsid)of47nucleotidesequencesof AstVs.(B)EvolutionarytreebasedonthepartialnucleotidesequencesofORF2from30sequencesofMAstV5.

acidlevels.ThepartialgenomesequencesofMAstV5obtained inthepresentstudyandthoseavailableinGenBank,together withselectedMamastrovirusreferencesequencesfromother species,generatedtwoevolutionarytrees(Fig.2).Forty-three referencestrainsandthefoursequencesfromthisstudy cor-respondingto19Mamastrovirusspeciesweredelineatedwith highbootstrapsupportthroughouttheentiretree(Fig.2A). In the MAstV5 clade, all of the present sequences clus-teredwith“Gillingham/2012/UK”(GenBankaccessionnumber NC026814), although with low amino acid identities of approximately80% (Table2).Consequently, thesefour new sequencesgroupedwithinChinesesequences,suggestinga differentgenotype putatively namedas MAsTV5a (Fig. 2B). Through a pairwise comparison of the ORF2 nucleotide sequence,ahighdegreeofnucleotideidentity,rangingfrom 95%to99%,wasdetectedamongthe Braziliantypestrains ofthisstudy.Thestudysequencesshowedacloser relation-shipwiththeChinesestrainsgroupingingenotypea.More distantstrainswereobservedamongtheHUN/2012/126 (Gen-BankaccessionnumberKX599352)Hungarystraincomposing thegenogroupb,Bari/2008ITA(GenBankaccessionnumber

HM045005) and Gillingham/2012/UK (GenBank accession numberNC026814)strainsfromItalyandUnitedKingdom, respectively,groupinginthegenotypec,andfinally,forming the genotype d, HUN/2012/115 (GenBank accession num-ber KX599351), HUN/2012/126 (GenBank accession number KX599352)strainsfromHungaryandGI.E/Dog/ITA/2010/Zoid (GenBankaccessionnumberJN193534)strainfromItaly.This suggestsadistinctionoffoursub-lineagesamongtheMAstV5 species–MAstV5atoMAstV5d(Fig.2B).

Discussion

Here, in a screeningof dog fecal samples, 26% (71/269) of thedogswithandwithoutdiarrheawereMAstV5positive,as determinedusingRT-PCR.Likewise,non-viralagentsand fac-torssuchasbacteria,intestinalparasites,malnutrition and intoxicationsareabletopromoteentericdiseasemainlyinthe youngdogpopulation.Thesearchforotherentericvirusesin theMAstV5-positivesamplesfromdogswithgastroenteritis showedthatthe dogswerealsoinfectedwithother known

pathogens.Moreover,wefoundthatsingleMAstV5infection wasassociatedonlywiththeasymptomaticstate,although thereisariskthattheresultswillbebiased,sincetheanalyzes wereconductedonthebasisofconveniencesamplingandwe cannotexcludethepossibilitythatthelongtermofviral shed-dingcouldbeanexplanationfortheMAstV5-positivesamples detectedinasymptomaticdogs,basedonpreviousstudythat demonstratedthecomparisonbetweenvirusloadandclinical manifestation26_{(Fig.1andTable3).Thesefindingswerenot}

unexpected,asmixedinfectionsarecommon,butmore stud-ieswillbenecessarytorealdeducetheroleofMAstV5inthe casesreportedhere.15–24

Several reports of MAstV5 suggest a clinical associa-tion ofvirusmolecular detection anddiseased dogclinical samples.18,20–23,26,33 _{Furthermore, studies of the prevalence}

ofMAstV5 inChina showed that 12% (22/183) of the pup-pies displaying clinical signs of diarrhea were positive for MAstV5,asdeterminedusingRT-PCR,comparedtononeof 138healthydogs,althoughthesestudiesdidnotlookforother virusesthatmaybeassociatedwithdiarrhea.19Inastudy con-ductedinItaly,24%of110stoolsamplescollectedfromdogs withclinicalsignstestedpositiveforthepresenceofMAstV5 RNA, and 9%(10/110) ofthe samples showed an MAstV5-singleinfection,althoughother asymptomatic animals(9% of75)were alsopositiveforMAstV533_{.Therefore,the}

asso-ciationwithclinicalsignsandthesheddingoftheviruswas describedonlyinacasestudyof2animals,whichis appar-entlyanisolatedcase.26_{AprevalencestudyinFrancefound}

that21%(66/316)ofthepuppiesin42%(14/33)ofthe breed-ingkennels surveyedwereMAstV5 positive,asdetermined usingRT-PCR.21_{Inthesamereport,theauthorsobservedthat}

puppiesthatwerelessthan7weeksoldwereespecially sus-ceptibleto MAstV5infection, althougha directassociation withclinical signswasnotpossible.21 _{Lastly,recentstudies}

foundaMAstV5prevalenceof6%intheUnitedKingdomand aninfectionrate of33% inpuppiesunderthreemonthsin Japan.22,23

Thepartialgenomic sequencingand characterizationof selectedsamplesrevealedaremarkablegeneticheterogeneity ofMAstV5ofBrazilianorigin.Becauseitwashypothesizedthat twostrainsofhumanAstVwithlessthan95%identityatthe nucleotidelevelareserologicallydistinguishable,34_thelower

identities(<85%)shownbetweenthecapsidgenesequences analyzed here may reflect the need for a novel species classification into four genotypes – MAstV5a to MAstV5d. Additionally, phylogenetic analysis indicated that the four MAstV5strainsreportedhererepresentalineagethatismore closely related to the Chinese strains than to the others strains,basedonthehighsequenceidentity(97%)ofORF2, accordingtothespeciesdemarcationcriteriaestablishedby

theICTV5_(Table2).

Insummary,wefound26%MAstV5-positivefecalsamples indogswithorwithoutgastroenteritis.Basedonsequence analysis of the partial genome from four MAstV5-positive samples,weproposedanovelspeciesclassificationintofour genotypes–MAstV5atoMAstV5d.Morestudiesarerequired tounderstandthebiologyandattempttheclinicaland anti-genicimplicationsofastrovirusgenotypesindogstoelucidate therelative veterinaryimportanceofdifferent canineAstV types.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorswouldliketoexpresstheirgratitudetotheclinical practitionerswho generouslyprovidedsamplesforanalysis andtothegraduateandpost-graduatestudentsofthe Labo-ratóriodeVirologiafortheirexcellenttechnicalsupportinthis work.

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