(genotype I) virus in Pune, India

19 Pages·2017·3.9 MB·English

RESEARCHARTICLE
Co-circulation of all the four dengue virus
serotypes and detection of a novel clade of
DENV-4 (genotype I) virus in Pune, India
during 2016 season
ShubhamShrivastava1,DivyaTiraki1,ArundhatiDiwan2,SanjayK.Lalwani3,
MeeraModak4,AkhileshChandraMishra1,VidyaA.Arankalle1*
1 DepartmentofCommunicableDiseases,InteractiveResearchSchoolforHealthAffairs,Bharati
VidyapeethDeemedUniversity,Pune,Maharashtra,India,2 DepartmentofMedicine,BharatiVidyapeeth
a1111111111
DeemedUniversityMedicalCollege,Pune,Maharashtra,India,3 DepartmentofPediatrics,Bharati
a1111111111
VidyapeethDeemedUniversityMedicalCollege,Pune,Maharashtra,India,4 DepartmentofMicrobiology,
a1111111111
BharatiVidyapeethDeemedUniversityMedicalCollege,Pune,Maharashtra,India
a1111111111
a1111111111 *[email protected]
Abstract
OPENACCESS Dengueisthemostcommonmosquito-borneviralinfectionintropicalandsub-tropicalcoun-
Citation:ShrivastavaS,TirakiD,DiwanA,Lalwani tries.Inrecentyears,Indiahasreportedincreasedincidencesofconcurrentinfectionwith
SK,ModakM,MishraAC,etal.(2018)Co- multipleserotypesofdengueviruses(DENV).Inthepresentstudy,wehavecharacterized
circulationofallthefourdenguevirusserotypes
DENVcirculatingduringasingleseasonof2016inPune,India.Atotalof64serumsamples
anddetectionofanovelcladeofDENV-4
(genotypeI)virusinPune,Indiaduring2016 fromNS1ELISApositivedenguepatientswereusedforPCRamplificationofCprMregion
season.PLoSONE13(2):e0192672.https://doi. oftheviralgenomeandsequencing.Phylogeneticanalysisdocumentedcirculationofallthe
org/10.1371/journal.pone.0192672
fourDENVserotypeswithpredominanceofDENV-2(40.6%).DENVgenotypingclassified
Editor:AftabA.Ansari,EmoryUniversitySchoolof DENV-1toGenotypeV,DENV-2toGenotypeIV,DENV-3toGenotypeIIIandDENV-4to
Medicine,UNITEDSTATES
GenotypeI.Furtheranalysisrevealedemergenceofanovelclade(D)ofgenotypeIof
Received:November18,2017 DENV-4.SubsequentisolationofthreeDENV-4virusesincellculturefollowedbycomplete
Accepted:January22,2018 genomesequenceanalysisconfirmedthisobservation.Additionally,anewgenotypewithin
serotype-4with>6.7%sequencevariationfromothergenotypeswasidentified.Thisfirst
Published:February22,2018
reportofsignificantco-circulationofallthefourserotypesinasingleoutbreakinPunerecon-
Copyright:©2018Shrivastavaetal.Thisisan
firmsneedformolecularmonitoringofDENV.
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Introduction
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
Anestimated40%oftheglobalpopulation(~3.9billion)isatriskofdenguevirus(DENV)
files.
infection[1,2].About2.5%ofpeopleaffectedwithseveredenguedieeachyear[3].Thedis-
Funding:ThisworkissupportedbyIndianCouncil easeisendemicinmorethan125countriesandthespreadtonewerareasismainlyattributed
ofMedicalResearch(ICMR)grantnumber:ECD/
toreturningtravelersfromendemiccountries[4,5].TherearefourserotypesofDENV
NTF/8/2016-17receivedbyACM.Thefunderhad
(DENV-1to-4)andallofthemcancausedenguefever(DF),aself-limitingfebrileillness.A
noroleinstudydesign,datacollectionand
variableproportionofpatientsprogresstolifethreateningdenguehemorrhagicfever(DHF)
analysis,decisiontopublish,orpreparationofthe
manuscript. characterizedbythrombocytopeniaandhemorrhage,anddengueshocksyndrome(DSS)due
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Co-circulationofdenguevirusesinPune,India
Competinginterests:Theauthorshavedeclared toexcessiveplasmaleakage[6,7].DENVhasbeenincirculationintheIndiansubcontinent
thatnocompetinginterestsexist. since1950s[8].ThefirstvirologicallyprovenepidemicofDFoccurredinKolkatain1963–
1964andatpresentthevirushasspreadto35statesandunionterritoriesinthecountry
(NVBDCP,http://nvbdcp.gov.in/den-cd.html)[9].
Onaccountofsequencevariability,dengueserotypesarefurtherclassifiedintodistinct
genotypesthatdiffer>6%withinasingleserotype[10–12].Emergenceofnewserotypeorline-
age\cladeshiftsincirculatingDENVgenotypesledtoenhancedseverityduringdengueout-
breaks[13–17].AlineageshiftinDENV-3wasreportedtocauseseverediseaseinSriLanka
[13,18].EmergenceofgenotypeIIIofDENV-3in2005resultedindengueoutbreakinNorth-
ernIndia[19].Recently,emergenceofAsianorgenotypeIofDENV-1alsocausedlargeout-
breakofdenguewith12,000casesinTamilNadu,SouthIndia[20].
AllthefourserotypesofDENVhavecirculatedinIndiaatdifferenttimes,butgenerallyone
serotypedominatesagivenoutbreak.Dengueoutbreakin1996inDelhiwascausedbygeno-
typeIVofDENV-2replacinggenotypeVisolatesof1957and1967[21]andvirusremainedin
circulationtill2002.Secondoutbreakin2003inDelhiwasduetoemergenceofDENV-3
whichremainedasdominantserotypetill2006[19].Overaperiodfrom2007–2009,DENV-1
becamethepredominantserotypeinDelhibyreplacingDENV-2andDENV-3[22].Earlier
dengueoutbreakswereattributedtosuddenemergenceofserotypeorgenotypethatco-circu-
latealongwithexistinggenotypeforsometimebeforegettingreplacedbyothersinsubsequent
years.Inrecentyears,co-circulationofmultipleserotypeshasbeenreportedfromdifferent
partsofIndia[23].Highpercentageofco-infectionwithmorethanoneserotypewasalso
observedwithincreaseddiseaseseverity[24–26].In2017,co-circulationofallfourDENV
serotypesinsingleoutbreakwasreportedfromOdisha[27]andHyderabad[28].
Punecity,westernIndiawithapopulationof112million(census2011)isendemicforden-
gue[29].Inviewofthepossibilityofintroductionofdenguevaccineinnearfuture,itisessen-
tialtounderstandthetypeandproportionofcirculatingDENVstrains.Thepresentstudy
reportsmolecularcharacterizationofdenguevirusescirculatinginPuneduringthe2016-den-
gueseason.
Methods
Samplecollection
Patientspresentingwithdenguelikesymptomsfor<4daystotheMedicineandPediatric
OPDsoftheBharatihospital,atertiarycarehospitalfromPunewereincludedinthestudy.To
avoidsecondprick,consentfortheuseofbloodsamplefordenguemolecularstudieswas
obtainedfromallthesuspectedpatients.Thisincludedwritteninformedconsentfromthe
parents(subjectsbelow7yearsofage),writteninformedassentandconsent(subjectsand
theirparentsrespectively,agegroup7–17years)andwritteninformedconsent(subjectsabove
17yearsofage).NS1positive(DengueEarlyELISA,Panbio,Windsor,Qld,Australia),leftover
serumsamples(n=120)werecollectedfromthediagnosticlaboratoryofthehospital.The
studywasapprovedbyInstitutionalEthicsCommittee,BharatiVidyapeethDeemedUniver-
sity,PunewithapprovalnumberIEC/2017/04.
Virusisolation
Onedaypriortoinfection,1x104Verocellswereseededineachwellof96-wellplateand
incubatedat37˚Cin5%CO incubator.100μlof10-foldseriallydilutedpatient’sserumin
2
quadruplatewellsin96-wellplatewasusedtoinfectVerocellsgrowninMinimumEssential
Medium(MEM,Gibco,ThermoScientific)containing2%fetalbovineserum,1%penicillin
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Co-circulationofdenguevirusesinPune,India
andstreptomycin.7dayspostinfection,culturesupernatantwasharvestedfromNS1positive
wellsandaliquotswerestoredat-80˚C.
ViralRNAextraction
TotalRNAwasextractedfrom140μlofhumanserumorcellcultureisolatesusingaQIAmp
viralRNAkit(QIAGEN,INC,Valencia,CA),aspermanufacturer’sprotocol.RNAwaseluted
in50μlofAVEbufferprovidedwiththekit.Foraconventionalgel-basedPCR,aminimumof
onenegativecontroleveryfoursampleswithnopresenceoftargetRNAwasincludedasapart
oftheextractionprocedure.
cDNAsynthesis
DenguespecificviralRNAwasreversetranscribedandamplifiedforCprMregionoftheviral
genomeasreportedbyChienetal(2006)[30].Forthis,single-strandedcDNAwassynthesized
fromtotalRNAusingthehighcapacitycDNAreversetranscriptionkit(Invitrogen).Briefly,
10μloftheextractedRNAwasaddedtothe2XRTmastermixconsistingof2μlof10XRT
buffer,0.8μlof100mMdNTPmix,2μlofreverseprimerD2(TTGCACCAACAGTCAATGTC
TTCAGGTTC-616)and1μlofMultiScribereversetranscriptase.Thereactionwasthensub-
jectedtoreversetranscriptionat25˚Cfor1min,37˚Cfor120min,85˚Cfor5min.Thepre-
paredcDNAwasimmediatelyusedorstoredat-20˚Cuntiluse.
PCRandsequencing
CprMregionwasPCRamplifiedusingAmpliTaqpolymerasekit(Invitrogen).5μlofthesyn-
thesizedcDNAwasthenaddedtothePCRmixcontaining10μlofPCRbuffer,10μlofMgCl ,
2
5μlofprimersmD1(134-TCAATATGCTGAAACGCGAGAGAAACCG)andD2each,0.5μl
dNTPs,1μlofpolymerase.Thereactionmixturewasthensubjectedto35cyclesofdenatur-
ationat94˚Cfor1min,annealingat55˚Cfor1min,andextensionat72˚Cfor1min.Theprod-
uctswerethenvisualizedfor511bpbyethidiumbromideagarosegelstaining[30].Amplified
productswerethenextractedfromthegelsusingQiaquickGelextractionkit(QIAGEN,INC,
Valencia,Calif)andbothstrandsweresequencedbyusingaBigDyeTerminatorCycle
Sequencingkit(AppliedBiosystems).TheCprMsequenceswereconfirmedbyBLAST(www.
ncbi.nlm.nih.gov/BLAST).Theforwardandreversesequenceswerealignedandmanually
editedusingCodonCodealignerv.7.0.1softwaretoobtaintheconsensussequence.Newpar-
tialCprMsequencesweresubmittedtoGenBankatwww.ncbi.nlm.nih.gov/genbank(acces-
sionnumberMG053110-MG053173).
Completegenomesequencing
FullgenomesequencingofviralgenomeswasdoneusingIonProtonsystem(Lifetechnolo-
gies,USA).Briefly,productswerepurified,sizeselected,amplifiedandquantified.Clonal
amplificationwascarriedoutbyemulsionPCRandtheIonsphereparticlesweredepositedon
toIonPIchip.Allprotonquality-approved,trimmedandfiltered(againsthumangenome)
datawereexportedasBAMfilesforbioinformaticsanalysis.Unmappedreadswerequalityfil-
teredwithmeanqualityscore>=20,minimumlength20andtrimmedusingPrinSeq-Lite
program.ResultinghighqualityreadswereassembledusingMIRAv4.0.2assemblerandcon-
tigswereannotatedusingBLASTagainstNCBIdatabase.Completegenomesequenceswere
submittedtoGenBankatwww.ncbi.nlm.nih.gov(accessionnumberMG272272-MG272274).
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Co-circulationofdenguevirusesinPune,India
Phylogeneticanalysis
ThesequencesobtainedinthepresentstudyandothersequencesretrievedfromGenBank
werealignedusingMAFFTonlinealignmenttool[31].Phylogenetictreeswereconstructed
usingMaximumLikelihoodmethodbasedonTamuraNeimodelinMEGA6.06software
[32].Geneticdistanceswerecalculatedusingthep-distancemodelofnucleotideandamino
acidsubstitution.Therobustnessoftheresultingtreewasassessedwith1000bootstrap
replicates.
Results
Patientcharacteristics
During2016-dengueseason,serumsamplesfrom109NS1positivepatientsweresubjectedto
RT-PCRand53(48.6%)scoredpositiveforDENV-RNA.Further,11cellculture-grown
DENVisolatesobtainedfromadditionalNS1positivepatientsweresubjectedtoRT-PCR.Of
the64patients,ageofthepatientsrangedfrom5monthsto65yearswithmedianageof28.6
years.Male(n=35)tofemale(n=29)ratiowas1:0.8.BasedonWHO2009guidelines,
63patientswerecategorizedasdengueillnessofwhich59withoutwarningsignsand4with
warningsigns.Onepatientwasclassifiedasseveredengue.DetailsareprovidedinTable1.
DENVserotypedistribution
Fig1depictsCprMgenephylogeny-basedserotypingof64DENVsequencesobtainedduring
thisstudy.Clearly,allthefourserotypeswerecirculatinginPuneduringthe2016season.Of
Table1. DemographicsandclinicalparametersofpatientsinfectedwithDENVwithdifferentserotypes.
Sr.No. SampleNo. Sampletype NS1Detection Age Gender ClinicalManifestation GenBankAccessionNo.
DengueSerotype1andgenotypeV
1 S44 Serum pos 36 M DwoWS MG053110
2 S58 Serum pos 32 F DwoWS MG053111
3 S59 Serum pos 18 M DwoWS MG053112
4 S105 Veroisolates pos 27 F DwoWS MG053113
5 S5 Serum pos 10 F DwoWS MG053114
6 S19 Serum pos 24 M DwoWS MG053115
7 S16 Serum pos 10 M DwoWS MG053116
8 S10 Serum pos 4 M DwoWS MG053117
9 S51 Serum pos 47 M DwoWS MG053118
DengueSerotype2andgenotypeIV(Cosmopolitan)
10 141 Serum pos 30 M DwoWS MG053119
11 812 Serum pos 26 F DwoWS MG053120
12 815 Serum pos 22 M DwoWS MG053121
13 892 Serum pos 60 F DwoWS MG053122
14 1008 Serum neg 21 F DwoWS MG053123
15 1053 Serum pos 20 M DwoWS MG053124
16 1571 Veroisolates pos 25 F DwoWS MG053125
17 S107 Veroisolates pos 16 M DwoWS MG053126
18 S47 Serum pos 25 M DwoWS MG053127
19 S57 Serum pos 24 M DwoWS MG053128
20 S77 Serum pos 5month M DWS MG053129
21 S85 Serum pos 11 F DWS MG053130
22 S94 Serum pos 27 M DwoWS MG053131
23 S87 Serum pos 35 M DwoWS MG053132
(Continued)
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Table1. (Continued)
Sr.No. SampleNo. Sampletype NS1Detection Age Gender ClinicalManifestation GenBankAccessionNo.
24 S25 Serum pos 25 F DwoWS MG053133
25 S26 Serum pos 26 M DwoWS MG053134
26 S52 Serum pos 34 F DwoWS MG053135
27 S53 Serum pos 50 M DwoWS MG053136
28 S66 Veroisolates pos 41 M DwoWS MG053137
29 S67 Veroisolates pos 38 F DwoWS MG053138
30 S97 Veroisolates pos 22 M DwoWS MG053139
31 S100 Veroisolates pos 18 M DwoWS MG053140
32 S4 Serum pos 16 F DwoWS MG053141
33 S15 Serum pos 12 F DwoWS MG053142
34 S9 Serum pos 34 F DwoWS MG053143
35 S78 Serum neg 21 F DwoWS MG053144
DengueSerotype3andgenotypeIII
36 1389 Serum pos 19 F DwoWS MG053145
37 984 Serum pos 18 F DwoWS MG053146
38 S108 Serum pos 24 M DwoWS MG053147
39 S73 Serum pos 41 F DwoWS MG053148
40 S45 Veroisolates pos 26 F DwoWS MG053149
41 S33 Serum pos 25 F DwoWS MG053150
42 S111 Veroisolates pos 30 F DwoWS MG053151
43 S56 Serum neg 42 M DwoWS MG053152
44 S54 Serum pos 39 M DwoWS MG053153
45 S50 Veroisolates pos 13 M DwoWS MG053154
46 S74 Serum pos 20 M DwoWS MG053155
47 S112 Veroisolates pos 20 M DwoWS MG053156
48 S1 Serum pos 26 F DwoWS MG053157
49 S76 Veroisolates pos 8 M DwoWS MG053158
50 S81 Serum pos 10 M DwoWS MG053159
51 S82 Serum pos 41 M DWS MG053160
52 S65 Serum pos 18 F DwoWS MG053161
DengueSerotype4andgenotypeI
53 S28 Serum pos 55 M DwoWS MG053162
54 S30 Serum pos 42 M DwoWS MG053163
55 S46 Serum pos 37 M DWS MG053164
56 S80 Serum pos 21 M DwoWS MG053165
57 S2 Serum pos 16 F DwoWS MG053166
58 36 Serum pos 6 F SD MG053167
59 294 Serum pos 65 F DwoWS MG053168
60 1018 Serum pos 2 M DwoWS MG053169
61 1021 Serum pos 20 F DwoWS MG053170
62 1028 Serum pos 17 M DwoWS MG053171
63 S49 Serum pos 47 F DwoWS MG053172
64 S41 Serum pos 23 F DwoWS MG053173
DwoWS:Dengueillnesswithoutwarningsigns
DWS:Dengueillnesswithwarningsigns
SD:Severedengue
https://doi.org/10.1371/journal.pone.0192672.t001
these,DENV-1wasdetectedin9(14.1%,Pune-2016-DENV1),DENV-2in26(40.6%,Pune-
2016-DENV2),DENV-3in17(26.6%,Pune-2016-DENV3)andDENV-4in12(18.7%,Pune-
2016-DENV4)samples.Thus,DENV-2wasfoundtobethepredominantserotypeanda
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Co-circulationofdenguevirusesinPune,India
Fig1.PhylogeneticanalysesofCprMgenesequencesfrom64DENVpositivecasesforserotypedetermination.
EachstrainisidentifiedbyGenbankaccessionnumberfollowedbycountryandyearofisolation.Numbersatthe
nodesaresupportvaluesforthemajorbranches(bootstrap;1000replicates).Thesequencesobtainedinthisstudyare
markedinfilledcoloredcircles.Scalebarindicatesnumberofbasesubstitutionspersite.
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Co-circulationofdenguevirusesinPune,India
Fig2.GenotypinganalysesofCprMgenesequencesofDENV-1serotypeisolates(n=9)fromPune.Eachstrainis
indicatedbyGenbankaccessionnumberfollowedbycountryandyearofisolation.Numbersatthenodesaresupport
valuesforthemajorbranches(bootstrap;1000replicates).Thesequencesobtainedinthisstudyaremarkedinfilled
coloredcircles.Scalebarindicatesnumberofbasesubstitutionspersite.
https://doi.org/10.1371/journal.pone.0192672.g002
substantialproportionofpatientswereinfectedwithotherserotypesaswell.Asfarasserotypic
distributionamongdifferentclinicalformsisconsidered,theonlyseveredenguepatientwas
infectedwithserotype4,patientswithdengueillnesswithoutwarningsignswereinfectedwith
eitherof4serotypesandthosewithwarningsignswereinfectedwithserotypes2,3or4.
DENVgenotypedistribution
TodeterminethegenotypedistributionofDENVwithineachserotype,CprMgenesequences
obtainedduringthisstudyandsequencesfromdifferentgeographicallocationsacrossthe
globewereretrievedfromNCBIdatabaseandusedforphylogeneticanalyses(Figs2–5).
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Fig3.GenotypinganalysesofCprMgenesequencesofDENV-2serotypeisolates(n=26)fromPune.Eachstrain
isindicatedbyGenbankaccessionnumberfollowedbycountryandyearofisolation.Numbersatthenodesare
supportvaluesforthemajorbranches(bootstrap;1000replicates).Thesequencesobtainedinthisstudyaremarkedin
filledcoloredcircles.Scalebarindicatesnumberofbasesubstitutionspersite.
https://doi.org/10.1371/journal.pone.0192672.g003
ForDENV-1strains,phylogenetictreerevealedclusteringofDENV-1sequencesintosix
genotypes.ThePune-2016-DENV1isolates(n=9)groupedintoAmerican/African(AM/AF)
orgenotypeVtogetherwithotherIndianisolatesfrom1962to2011(Fig2).Asreportedearlier
(Ceciliaetal,2017),oneisolatefromKerala,2013(KJ755855)belongedtoAsianorgenotypeI.
ThePune-2016-DENV1sequencesweresimilarwith99.8±0.3%nucleotideidentities.The
currentsequencesclusteredwithisolatesfromIndia(2008–2016),Singapore(2011–15),China
(2014)andBrunei(2005).
AsevidentfromFig3,DENV-2sequenceswereclassifiedintosixgenotypes.Pune-2016
sequences(n=26)groupedtogetherinCosmopolitanorgenotypeIVandexhibited99.3±
0.3%nucleotidesimilarity.Thisgenotypeisdividedintotwogeographicallydistinctlineages,
lineageA(isolatesfromSoutheastAsia,ChinaandOceania)andlineageB(isolatesmostly
fromIndiansubcontinent).Pune-2016sequencesbelongedtolineageBandclusteredwith
strainsfromIndia(2008–12),Pakistan(2008–13),China(1999),Singapore(2013)andSri
Lanka(2003).
PhylogeneticanalysisclassifiedPune-2016-DENV3(n=17)CprMsequencesingenotype
III(Fig4)withnucleotidesequencesimilarityof99.3±0.3%.GenotypeIIIstrainsexhibitwide
geographicdistributionfromAsia,Caribbean,AmericasandEurope.Pune-2016sequences
werecloselyrelatedtotheotherisolatesfromIndia(2004–2016),China(2009,2013),Singa-
pore(2009),Pakistan(2008–09)andasingleisolatefromSenegal(2009)with99.4±0.3%
nucleotidesimilarities.AmongtheotherIndiaisolates,1984-isolate(KF955477)groupedinto
GenotypeIIwhileasingleisolatefromnorthernIndia(KC787098,2009)wasassignedto
genotypeIVandwasfoundtobecloselyrelatedtoDENV-3prototypestrainofPhilippines,
1956(M93130).
DENV-4,therareserotypeinIndiawaspreviouslyreportedin2003fromDelhi,and2007
fromHyderabadand2010fromKerala[33–35].InMaharashtrastate,lastreportofDENV-4
caseswasin1975fromAmalnerdistrictandlaterdetectedinPunein2009afteragapof30
years[36].PhylogeneticanalysisrevealedthatDENV-4sequenceshavebeengroupedinto5
genotypeswithaninter-genotypicsequencedivergenceof>6%[10–12,37,38].Ourdatadoc-
umentedthat(1)thePune-2016viruses(n=12)formedadistinctclusterwithingenotypeI
and(2)11sequencesearlierclassifiedelsewhere[39,40]asgenotypeIIconstitutedaseparate
cluster(Fig5)thatincludedisolatesmostlyfromEastandSoutheastAsiancountriessuchas
Japan,China,Taiwan,Indonesia,SingaporeandPhilippines.
WefurthercomparedpercentnucleotidedivergenceinCprMregionamongdifferentclus-
terswithingenotypeIanddifferentclustersconstitutinggenotypeswithinserotypeIV
(Table2).ThenovelclusterincludingcurrentPunestrainswas3.0±0.6%to5.6±0.8%diver-
gentwhencomparedtotheotherclusters/cladeswithingenotypeIandtentativelydesignated
ascladeD.DENV-4virusesisolatedin2007(EU652498-EU652499)and2010(JN882277)
fromtwosouthernIndianstatestogetherwithPune-2016sequencesbelongedtocladeD.The
distinctclusterofsequencesearlierclassifiedasgenotypeIIwas6.8%-10.2%differentfrom
theknowngenotypesI–V(Table2).Theseresultssuggestedthatthisclustermayrepresenta
novelgenotypeVI.
Forconfirmationoftheseobservations,fullgenomesequenceanalysiswasdone.We
obtainedcompletegenomesequencesofthreeDENV-4strains,isolatedinVerocells(acces-
sionnumberMG272272-MG272274).ThecompletegenomelengthsofPune-2016isolatesare
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