Virus isolation and genotype identification of human respiratory syncytial virus in Guizhou Province, China

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w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Virus

isolation

and

genotype

identiﬁcation

of

human

respiratory

syncytial

virus

in

Guizhou

Province,

China

Ting

Zhao

a,b,∗

_,

_Zhixu

_Ye

c,d

_,

_Binlin

_Wang

e

_,

_Yuxia

_Cui

c

_,

_Yingjie

_Nie

b,d

_,

Bin

Yang

b,d

_,

_Kun

_Chen

b,d

_,

_Hua

_Zhang

f

_,

_Fangfang

_Hu

f

_,

_Fuxun

_Yu

b,d a_Guizhou_University,_Medical_College,_Guiyang,_China

b_Guizhou_University,_Guizhou_Provincial_People’s_Hospital,_Department_of_Central_Laboratory,_GuiyangChina c_Guizhou_University,_Guizhou_Provincial_People’s_Hospital,_Department_of_Pediatrics,_Guiyang,_China

d_Guizhou_Provincial_People’s_Hospital,_NHC_Key_Laboratory_of_Pulmonary_{Immunological}_Diseases,_Guiyang,_China e_People’s_Hospital_of_QianNan,_Department_of_Clinical_Labotatory,_Guizhou_Province,_China

f_Guizhou_University,_Guizhou_Provincial_People’s_Hospital,_Department_of_Clinical_Laboratory,_Guiyang,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24June2019

Accepted6October2019

Availableonline14November2019

Keywords:

Humanrespiratorysyncytialvirus

Virusisolation RT-PCR HVR2 Phylogeneticanalysis Molecularepidemiology

a

b

s

t

r

a

c

t

ToinvestigatethegeneticvariationandmolecularepidemiologycharacteristicsofHuman

RespiratorySyncytialVirus(HRSV)inGuizhouProvince,nasopharyngealaspirateswere

col-lectedfrompatientswithacuterespiratoryinfection(ARI)inGuizhouProvincialPeople’s

Hospital,fromDecember2017toMarch2018,andinoculatedtoHep-2cellstoisolateHRSV.

Cellsthatshowedcytopathiceffect(CPE)werethenconﬁrmedbyindirect

immunoﬂuores-cenceassayandreversetranscription.ThesequenceofthePCRproductswasdetermined

forHRSVisolates,andthegeneticvariationwasanalyzed.Outof196nasopharyngeal

aspi-ratesamples,HRSVwereisolatedin39.Thesecondhypervariableregionatthe3terminal

ofglycoprotein gene(HVR2)sequenceanalysisshowedthatsubgroupAwasdominant.

Seventy-ninepercentofthe isolatesbelongedto subgroupA, ON1genotype,and21 %

belongedtosubgroupB,BA9genotype,whichindicatesthatthedominantHRSVcirculating

inGuizhouProvincewassubgroupA,genotypeON1,co-circulatingwithalessprevalent

subgroupB,genotypeBA9.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Abbreviations: HRSV, Human respiratory syncytial virus;ARI, Acute respiratory infection; CPE,Cytopathic effect; IFA, Indirect

immunoﬂuorescenceassay;RT-PCR,Reversetranscriptionpolymerasechainreaction;HVR2,Thesecondhypervariableregion.

∗ _{Corresponding}_authors.

E-mailaddress:1577065205@qq.com(T.Zhao).

https://doi.org/10.1016/j.bjid.2019.10.007

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Introduction

Human Respiratory Syncytial Virus (HRSV) is one of the

most important pathogens causing acute respiratory tract

Infections(ARI)ininfants,elderly,andimmunocompromised

peopleworldwide.1,2 _HRSV_infection_is_common,_with_more

than 90 %ofchildreninfectedbefore the ageoftwo, with

0.5%–2% of cases requiring hospitalization due to severe

symptoms.3_In_2015,_Shi_et_al.4,5 _estimated_that_33.1_million

casesofHRSVARIoccurredinchildrenunderﬁve,including

3.2millionhospitalizationsand59,600in-hospitaldeaths.The

costsassociatedwiththeHRSVinfectionarehighduetolack

ofalicensedvaccineandspeciﬁctreatment.6

HRSV is a single-negative-strand RNA virus belonging

to the Paramyxoviridae family genus pneumoviridae.7 _The

genomicRNAofHRSVhasabout15,200nucleotidesinlength

containing10genes,whichareNS1,NS2,N,P,M,SH,G,F,M2,

andL,expressing11proteins.Amongthem,proteinGisone

oftheimportantantigensagainstwhichthehostoften

tar-getstoproduceprotectiveantibodies.However,theviruscan

escapehost’s immuneresponsethrough antigenmutation,

whichis animportantcause ofrecurrent HRSVinfection.8

HRSVisdividedintotwosubtypes,AandB;variabilityamong

thesesubtypesisconcentratedintwohighlyvariableregions

thatispartoftheextracellulardomainofproteinG.

Accord-ingtothesequenceofasecondhypervariableregion(HVR2)

withintheGgene,thetwogroupsofHRSVarefurtherdivided

intogenotypes.4,9 _In_recent_years,_the_most_common

geno-typesworldwidehavebeentheON1genotypeofsubgroupA

andtheBAgenotypeofsubgroupB.1,10–13

Guizhou Provinceis locatedinthe southeastern part of

southwest China, with jurisdiction over nine prefectures,

amongthemthreearetheminorityautonomousprefectures,

andthecapitalcityisGuiyang.GuizhouProvinceisa

multi-ethnicprovincewithatotalof56ethnicgroupswithethnic

minoritiesaccountingfor39%oftheprovince’stotal

popu-lation.Someoftheminoritiesliveinisolatedtribes.Henceit

isimportanttostudythemolecularepidemiccharacterand

theevolutionofHRSVinGuizhouProvince.Someprovinces

inChinahavereportedthegeneticvariationcharacteristicsof

HRSV,butGuizhouProvincelackssuchbasicdata.14–20_In_order

tounderstandtheHRSVinfectionsituationandthe

molecu-larepidemiccharacteristicsofHRSVinGuizhouProvince,we

carriedoutHRSVisolationfrompatientswithARIagedless

thanﬁveyearsoldinGuiyang,fromDecember2017toMarch

2018,andanalyzedtheHVR2sequenceoftheGgeneofthe

isolates.Thisstudyprovidesthebasicdatafortheprevention

andcontrolofHRSVinGuizhouProvince.

Methods

Clinicalsamples

Nasopharyngealaspirates were collected from hospitalized

children under ﬁve years old with ARI, mainly presenting

withbronchiolitisorpneumonia,inGuizhouProvincial

Peo-ple’sHospitalfromDecember2017toMarch2018.Thestudy

wasapprovedbytheEthicsCommitteesofGuizhouProvincial

People’sHospital(approvalnumber:2017105).

IsolationofHRSV

The nasopharyngeal aspirates were treated with penicillin

(100units/mL) and streptomycin (100␮g/mL) before being

inoculatedtoHep-2cells(ATCC)culturedinDulbecco’s

modi-ﬁedEagle’smedium(DMEM;GIBCO,CA)supplementedwith2

%fetalbovineserum(FBS;GIBCO).Thecytopathiceffect(CPE)

of inoculatedHep-2 cells was observedeveryday forseven

days.CPEnegativesamplesattheseventhdayafter

inocula-tionwereblindlyinoculatedfortwoconsecutivepassagesand

CPEpositivesamplesrecordedandstoredat−80◦_C_for_future

use.

Indirectimmunoﬂuorescenceassay

CPEpositiveanduninfected(mock)Hep-2cellsweredigested

by 0.02 % EDTA, spotted to eightwell glass slide(MP

Bio-chemicals, CA, USA)separately. Theslideswere ﬁxedwith

coldacetoneafterairdryingthecells.Afterblockingwith3%

BSAforonehouratroomtemperature,theslideswere

incu-batedwithsheepanti-HRSVIgGantibody(1:500,Millipore)at

37◦CforonehourfollowedbywashingthreetimeswithPBS,

the slideswere reacted with AlexaFluor 488labelled

anti-sheepIgG(1:500,Invitrogen)atroomtemperatureforonehour.

AfteradditionalthreewasheswithPBS,theslideswereair

dried,sealedwithmountingmediumforﬂuorescence(Vector

Laboratories,Inc.).Thesamplesweresubsequentlyexamined

usingaﬂuorescencemicroscope.

Reversetranscription,PCRandsequencinganalysis

Total RNA was extracted from 500␮L of the viral isolates

cultureﬂuidwithTrizol(Invitrogen)accordingtothe

manu-facturer’sinstructions.21_Reverse_{transcription}_was_performed

at25◦Cfor10minand37◦Cfor60min,inareactionmixture

containing11␮LofRNA,4␮Lof5×FirstStrandBuffer

(Invit-rogen),1mLof100mMDTT,1␮Lof10mMdNTPMix,1␮Lof

RNaseinhibitor(TaKaRa),1␮Lofrandomprimersand1␮Lof

superscriptIII(Invitrogen)inaﬁnalvolumeof20␮L.

NestedPCRwas carriedout withtwosetsofprimersto

amplifythethreeprimeendofHRSV10Ggeneforallthe

iso-lates.FortheﬁrstampliﬁcationforwardSH1(5

CACAGTKACT-GACAAYAAAGGAGC) and reverse F164(5

GTTATGACACTG-GTATACCAACC) primers were used, followed by a second

ampliﬁcation with ABG490 (5

ATGATTWYCAYTTTGAAGT-GTTC)andF9AB(5CAACTCCATKRTTATTTGCC)primerpair.6

One␮Lofthereversetranscriptionreactionmixturewasused

forthefirst amplification,whereas1␮Lofthefirst

ampliﬁ-cationmixture wasused forthe second PCR. PCRreaction

mixturescontained10×LAPCRbuffer,1mMMgCL,2.5mM

ofeachdNTPMix,0.25␮MofeachprimerandﬁveunitsofLA

Taqpolymerase(TaKaRa).PCRconditionsfortheﬁrst

ampliﬁ-cationwere:94◦Cfortwominutes,34cyclesof:94◦Cfor30s,

55◦Cfor30sand72◦Cforoneminuteand20s.One␮Lofthe

ﬁrstampliﬁcationmixturewasusedforthesecondPCR,which

wasperformedusingthesamereactionmixtureat55◦Cbut

(3)

oneminute.Theampliﬁcationproductswereseparatedona

1%agarosegel.

GelpuriﬁedPCRproductswereusedtosetupthe

sequenc-ingreactionsusingtheABG490andF9ABprimerstheBigDye

Terminator v3.1 Cycle Sequencing Kit (ThermoFisher

Sci-entiﬁc, USA) according to the manufacturer’s instructions.

Sequencingandsequenceanalysiswereperformedona3730

GeneticAnalyzer(ThermoFisherScientiﬁc,USA).

Homologyanalysis

The obtained sequence alignments were performed using

CLUSTALW. The nucleotide sequences of subgroup A

and B viruses were conﬁrmed by using BioEdit software

(http://www.mbio.ncsu.edu/BioEdit/)and homologyanalysis

wasconductedusingDNASTARsoftware.22

Phylogeneticanalysis

Phylogenetic trees of the G protein were performed using

CLUSTALW.Thenucleotidesequences ofsubgroupAand B

viruseswereconﬁrmedusingBioEditsoftwareandhomology

analysiswasdoneusingDNASTAR.9 _Bootstrap _{probabilities}

werecalculatedafter1000iterationstoevaluateconﬁdence

estimatesand bootstrap valuesforviruseswere conﬁrmed

usingBioEditsoftware.Homologybetweengenotypeswere

estimatedusingtheMEGAp-distancemethod,which

calcu-latesproportion(p)ofnucleotidesitesatwhichtwosequences

beingcomparedaredifferent.6

Analysisofdeducedaminoacidsequences

Deduced amino acid sequences were generated using

ORFﬁnder(https://www.ncbi.nlm.nih.gov/orfﬁnder/)andthe

obtainedaminoacidsequenceswereanalyzedusing

DNAS-TARforhomologyanalysis.BioEditwasusedtocomparethe

aminoacidsequencesoftheHVR2withtheprototypestrain.

Results

HRSVisolationandidentiﬁcation

FromDecember2017toMarch2018,wecollected196

nasopha-ryngealaspiratessamplesfrompatientswithARIsinGuizhou

Province,forHRSVvirusisolationbyHep-2cellinoculation.

HRSVpositiveisolatesshowedtypicalCPE.Theinfectedcells

becameroundandfusedtogetherforminggiantcells(Fig.1A

andB).Outofthe196samples,weobtained39(19.90%)HRSV

isolates.IFAresultsshowedthatalltheisolateswerereactive

toanti-HRSVantibody(Fig. 1Cand D). RT-PCRresults

con-ﬁrmedthatthese isolateswerepositive forHRSV.ThePCR

productsoftheseisolatesweresentforsequencingandthe

resultsshowed31strains(79%)ofsubgroupAandeightstrains

(21%)ofsubgroupB.

Homologyanalysis

ThegenesequencesobtainedbysequencingthePCRproducts

wereconﬁrmedwithBioEditandwerecomparedwithA2and

Fig.1–Cytopathiceffectandindirectimmunoﬂuoresence testofHRSVisolatesonHep-2cells.A,MockHep-2cell;B, IsolateGZA17-01infectedHep-2cells;C,Indirect

immunoﬂuoresencetestofmockHep-2cell;D,Indirect immunoﬂuoresencetestofisolateGZA17-01infectedHep-2 cell.

BprototypestrainsCH18537byusingClustalWandBioEdit

software.ThesequencelengthsobtainedforsubgroupAand

subgroupBisolateswere516nucleotidesand505nucleotides,

respectively. The homology analysis was performed using

DNASTAR.

Between theprototypesstrain A2andthe Guizhou

sub-group A isolates, the rates of identity were 86.1%–87.9%

atthe nucleotide level and 79.6%–81.6% atthe amino acid

level. IdentitywithintheGuizhousubgroupAisolateswere

94.4%–100% and 87.7%–100%at thenucleotide leveland at

the aminoacid level, respectively(Figs. 2A,Band S1).The

ratesofidentitybetweentheGuizhouBisolatesandthe

pro-totypicgenotypeBstrainCH18537were 76.9%–83.8%atthe

nucleotideand61.1%–70.1%attheaminoacidlevel.Identity

withintheGuizhousubgroupBisolateswere88.7%–99.2%and

80.8%–98.2%atthenucleotidelevelandattheaminoacidlevel,

respectively(Figs.3A,BandS2).

Phylogeneticanalysis

ThesequencesoftheHVR2Ggenesfrom31subgroupAand

eightsubgroupBisolatesobtainedinthisstudyandsome

rep-resentativegenotypessequencesdownloadedfromGenBank

wereusedforphylogeneticanalysis.

The newly isolated 31 HRSV subgroup A strains were

discoveredbearinga 72-ntduplication whencomparedthe

sequence ofthe prototype virus (referencestrain M11486).

These resultsrevealed thatall 31 subgroupAviruseswere

clusteredintogenotypeON1,whichweremostcloselyrelated

to ON1 strains KX533701 KX533692, KX533588, KT765058,

JN257693,andKC731482fromChina(Beijing,Sichuan),Canada

andIndia,respectively(Fig.4A).

AllnewlyHRSVsubgroupBisolateswerefoundtocluster

intheBAgenotypeandhadthesignature60bpduplication

in the G proteinwhen aligned withprototype B reference

strain CH18537. The HRSV subgroup B isolates were most

(4)

Fig.2–ComparisonofhomologybetweentheGuizhou HRSVsubgroupAisolates3endofGgenenucleotideand aminoacidsequenceswiththatoftheprototypestrainA2 incoloredheatmap.A,comparisonofnucleotide

sequenceshomologyofsubgroupA;B,homology comparisonofaminoacidsequencesofsubgroupA.Red indicatesthemaximumidentity,blueindicatesthe minimumidentity,whiteindicatesthemiddlevalue,green indicatesthemaximumdivergence,redindicatesthe minimumdivergence,yellowindicatesthemiddlevalue.

KT765101, and HM459879 from China (Hunan, Shanghai,

SichuanandChongqing)andJapan,respectively(Fig.4B).

Analysisofdeducedaminoacids

The predicted amino acid sequences of HVR2 G gene

sequencesdeducedfromthe270nucleotidefragmentofthe

subgroupAandsubgroupBisolateswereusedtogenerate

a consensus sequence atamino acid level for each

geno-typeincomparisonwithprototypestrainsA2andCH18537,

respectively. When compared with A2 strain, the newly

isolatedHRSVsubgroupAstrainswere ON1genotype with

theinsertionof24aminoacidsinHVR2region(Fig.5A).The

newlyisolatedsubgroupBstrainsobtainedinthisstudyhada

Fig.3–ComparisonofhomologybetweenGuizhouHRSV subgroupBisolates3endofGgenenucleotideandamino acidsequenceswiththatoftheprototypestrainCH18537in coloredheatmap.A,comparisonofnucleotidesequences homologyofsubgroupB;B,homologycomparisonof aminoacidsequencesofsubgroupB.Redindicatesthe maximumidentity,blueindicatestheminimumidentity, whiteindicatesthemiddlevalue,greenindicatesthe maximumdivergence,redindicatestheminimum divergence,yellowindicatesthemiddlevalue.

duplicationof20aminoacidsinHVR2regionwhencompared

withCH18537strain(Fig.5B).

Discussion

To investigate HRSV molecular epidemic characteristics in

Guizhou Province, we isolated 39 HRSV strains from ARI

patientsinGuizhouProvincefromDecember2017toMarch

2018.Theseisolateswere conﬁrmedtobeHRSVbyIFAand

RT-PCR. Thegenotypes oftheseisolates were identiﬁedby

sequenceanalysis.ThisistheﬁrstreportofHRSVvirus

iso-lationandgenotypinginGuizhouProvince,China.

Thevirus isolation ratewas lowerin ourstudy (19.9%)

(5)

Fig.4–PhylogenetictreesofnucleotidesequencesfromthesecondvariableregionoftheGgeneofHRSVsubgroupA(A) andsubgroupB(B)isolates.TreesweregeneratedbyusingtheNeighbor-joiningmethods.Bootstrapvaluesgreaterthan70 %areshownatthebranchnotes.Thevaluerepresentsthebootstrapprobability.Thegenotypesobtainedbyisolatesinthis studyareindicatedbybracketsontherightside.Referencesequencesdescribingeachgenotype(ON1,NA1toNA4,GA1to GA7,SAA1,GB1toGB4,SAB1toSAB3,BA1toBA10)wereobtainedfromtheGenbankdatabaseandincludedinthetree. Prototypestrains(strainA2forsubgroupAandstrainCH18537forsubgroupB)wereusedastheoutgroupsequences.

thenasopharyngealaspiratesampleusedforvirusisolation

wasthe remainingfromclinical bacteriaculturetests. The

amountleft forvirus isolation wassometimesinsufﬁcient.

Anotherreasonmaybebecauseweusedonlyonecellline

(Hep-2)forvirusisolation.Toimprovethevirusisolationrate

infuturestudies,usingseveralcelllinesforviruscultureand

freshclinicalsamplesmaybenecessary.Comparedwiththe

studiesofdirectPCRampliﬁcationusingextractedRNAfrom

nasopharyngealsampleswithoutvirusisolation,thepositive

rateofdirectPCRwasmuchhigher,indicatingthatgene

detec-tionmethodsweremuchmoresensitive.24,25RussoMetal.24

reportedthatout of520 samples,238werepositivebyreal

timeRT-PCR(positiverate55.5%).ThepositiverateofHRSV

was46.11%in180clinicalsamplesusingRT-PCRinanother

report.25

TheepidemicpatternofHRSVsubtypevariesfromregion

to region. In a given year, subtype A and subtype B may

co-exist in a region, or one of them may be dominant,

althoughsubgroupAinfectionisdominantinmostpartsof

the world.26 _For _example, _in_the _six _consecutive _epidemic

seasonsfrom2000to2006inBeijingarea,subgroupA

domi-natedin2001–2004,subgroupBdominatedin2004–2005,and

co-dominancebytwosubgroupsinthe2005–2006epidemic

season.27_A_total₅₄₈₃_children_with_respiratory_diseases_were

tested inGuangzhou city forthreeyears.Both subgroupA

positivecases(51.2%,373/729)andsubgroupBpositivecases

(48.8 %,356/729) werefound. SubgroupBwasdominantin

2013,subgroupAwasdominantin2015,andsubgroupAand

subgroupBweretransmittedtogetherin2014.28In2010–2014,

therewere570casesofHRSVinfectioninMexico,ofwhich

407casesbelongedtosubgroupA,andonly27.54%were

sub-groupB6_;_In_Croatia,_between₂₀₁₁_and_2014,_subgroup_A_was

thedominanttype,accountingfor76%ofHRSVinfections.29

Between 2011and 2013,subgroup Awasreported tobe

dominantinsouthernBrazil,outof167detectedHRSVcases,

(6)

Fig.5–DeducedaminoacidalignmentofHVR2fromGuizhouHRSVisolatesshownrelativetothesequenceofprototype strains.A,deducedaminoacidalignmentofHVR2fromGuizhousubgroupAisolatesshownrelativetothesequenceof strainA2(acc.noM11486);B,deducedaminoacidalignmentofHVR2fromGuizhousubgroupBisolatesshownrelativeto thesequenceofstrainCH18537(Numberacc.noJX198143).Differentaminoacidsareshownindifferentcolors,identical residuesareindicatedbydots,dashesdesignategapscorrespondingtonucleotideinsertionsandasterisksindicatestop codons.

subgroupA accounted for76.7 % of the 73 casesof HRSV

infectioninSaoPaulo,Brazil.30

Inthis study,out ofthe total39 HRSVisolates,31 were

subgroupAandeightweresubgroupB.Indicatingthatboth

subgroupswereco-circulatinginGuizhouProvince,but

sub-groupApredominatedasseeninotherareasoftheworld.

Genotypeshifting ofHRSViscommon.From 2003–2008,

studieshaveshownthatthedominantgenotypeofsubgroupA

wasGA2insomeprovincesofChinawhilethedominant

geno-typeofGA2wasreplacedbygenotypeNA1.NA1genotypewas

ﬁrstreportedinBeijingfrom2008to2013.31_Similarly,_the_ON1

genotypewasﬁrstfoundinCanadain2010andhassincebeen

reportedintheUnitedStates,Latvia,SouthKorea,Thailand,

Japan, Kenya, West India, and other countries.32 _In _China,

theON1genotypewasﬁrstdetectedinShanghaiinFebruary

2011.33_Since_then,_ON1_genotypes_have_become_increasingly

common,replacingNA1genotypesasthedominantgenotype

inChina.31,33 _In _Brazil,_the _new_HRSV-A _genotype _ON1was

introducedin2012andbecameprevalentin2013.11Globally,

differentON1genotypeshavebeenreportedin21countries

andhaverapidlyspreadaroundtheworld.32_As_for_subgroup

B,theBAgenotypewasﬁrstreportedinTrentoin1999,then

detectedinJapanin2003,andsubsequentlyinSouthAfrica,

China,Brazil,andThailand,becomingthedominantsubgroup

Bgenotypeworldwide.34_In_China,_the_BA_strain_was_ﬁrst

iden-tiﬁed inBeijing in 2004and rapidly became the dominant

one.22

AlthoughON1andBAstrainsappeartohavebeenmore

successfulin replacing other genotypic strains, the reason

for their dominance is not clear. Some authors reported

thatthereisnoindicationthatON1and BAstrainsleadto

more serious disease,6,22 _while _others _reported _that _ON1

genotype showedan increasedtendency todevelopsevere

disease.11,35 _Among _the ₇₂ _nucleotide _duplication _in _ON1

genotype ofsubgroup A and the 60 nucleotideduplication

in BA9 genotype of subgroup B,some minor substitutions

at the nucleotide level and amino acid level were found.

Whethertheseminormutationscausethediversityreported

bydifferentresearchgroupsisnotclear.Moredetailedstudies

focusingonthevirologyandimmunologycharacteristicsof

differentgenotypesareneededtoillustratethemechanism

ofgenotypeshiftandthepathogenicityofthevirus.

Phylogenetic analysis revealed that all isolates of

sub-groupAinGuizhouProvincewereclassiﬁedasON1witha72

nucleotideduplicationintheHVR2gene,whileallsubgroup

BisolateswereclusteredtoBA9withtypicalduplicationof60

nucleotidesintheHVR2domain.Thisﬁndingindicatesthat

ON1genotypeinsubgroupAandBA9genotypeinsubgroup

B were predominantin Guizhou Province asseen in other

places.

Inconclusion,wereportedfortheﬁrsttimetheisolation

and identiﬁcationofHRSVinGuizhouProvince.The3 end

ofGgeneofthe39isolateswassequencedandanalyzed.In

GuizhouProvince,bothsubgroupAandBwerecirculatingin

2017–2018,butthemajorityofclinicalisolateswassubgroup

A,ON1genotype.AllsubgroupBisolateswereofBA9

geno-type.FurthersurveillanceofHRSVgeneticvariationisneeded

forpreventingandcontrollingofHRSVinfectioninGuizhou

Province.

Author

contributions

FY, YC, ZY and YN designed the study. TZ, BW, WH, LL,

ZL, BY, QX, KC, FH, HZ and FY performed the

experi-ments. TZ, ZY and FY analyzed the data. TZ and FY

wrote the paper. All authors read and approved the ﬁnal

(7)

Funding

ThisstudywassupportedbyFundoftheScienceand

Technol-ogyDepartmentofGuizhouProvince([2018]2785,[2015]4015,

[2017]5781,[2018]5706,[2018]5801),theNationalNatural

Sci-enceFoundationofChina(2018)81860003,(2019)81960001.

Ethics

approval

and

consent

to

participate

Thestudy was approved by the Ethics Committeesof the

ChinaGuizhouProvincialpeople’sHospital(Approvenumber

2017105).

Conﬂict

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

Theauthors wish to thankthe staff ofGuizhou Provincial

People’sHospitalfortheassistanceofsamplecollectionand

researchcooperation.WethankDr.KlarkeM.Samplefromthe

GuizhouProvincialPeople’sHospitalforeditingtheEnglishof

ourmanuscript.

Appendix

A. Supplementary

data

Supplementary material related to this article can be

found, in the online version, atdoi:https://doi.org/10.1016/

j.bjid.2019.10.007.

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