Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII.4 2012" variants in UK coastal waters during 2013

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

Environmental

Microbiology

Molecular

screening

of

blue

mussels

indicated

high

mid-summer

prevalence

of

human

genogroup

II

Noroviruses,

including

the

pandemic

“GII.4

2012”

variants

in

UK

coastal

waters

during

2013

Subhajit

Biswas

∗,1

_,

_Philippa

_Jackson,

_Rebecca

_Shannon,

_Katherine

_Dulwich,

Soumi

Sukla

1

_,

_Ronald

_A.

_Dixon

UniversityofLincoln,SchoolofLifeSciences,BrayfordPool,Lincoln,Lincolnshire,UnitedKingdom

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5April2017 Accepted22June2017

Availableonline13October2017 AssociateEditor:MauricioNogueira

Keywords:

Bluemussels Norovirus

GII.4Sydneyvariant Coastalwater

a

b

s

t

r

a

c

t

Thismolecularstudyistheﬁrstreport,tothebestofourknowledge,onidentiﬁcationof norovirus,NoVGII.4Sydney2012variants,frombluemusselscollectedfromUKcoastal waters.Bluemussels(threepooledsamplesfromtwelvemussels)collectedduringthe2013 summermonthsfromUKcoastalsiteswerescreenedbyRT-PCRassays.PCRproductsof

RdRPgenefornoroviruseswerepuriﬁed,sequencedandsubjectedtophylogeneticanalysis. AllthesamplestestedpositiveforNoVs.SequencingrevealedthattheNoVpartialRdRP

genesequencesfromtwopooledsamplesclusteredwiththepandemic“GII.4Sydney vari-ants”whilsttheotherpooledsampleclusteredwiththeNoVGII.2variants.Thismolecular studyindicatedmusselcontaminationwithpathogenicNoVsevenduringmid-summerin UKcoastalwaterswhichposedpotentialriskofNoVoutbreaksirrespectiveofseason.As thedetectionofSydney2012NoVfromourpreliminarystudyofnaturalcoastalmussels interestinglycorroboratedwithNoVoutbreaksinnearbyareasduringthesameperiod,it emphasizestheimportanceofenvironmentalsurveillanceworkforforecastofhighrisk zonesofNoVoutbreaks.

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

Introduction

Blue mussels (Mytilus spp) are ﬁlter-feeders and are

widespread in European coastal waters, which are often

in close proximity to urban sewage treatment units.1,2

∗ _{Corresponding}_author.

E-mail:subhajit.biswas@iicb.res.in(S.Biswas).

1 _Current_address:_CSIR-Indian_Institute_of_Chemical_Biology,_4,_Raja_S.C._Mullick_Road,_Kolkata,_West_Bengal_700032,_India.

Filter-feeders may retain particles at 4␮m with 100%

efﬁciency.3 _Faecal _{contamination} _of _water _(via _sewage

discharge)posesaseriousthreattohumanhealthasbivalves

(mussels and oysters) which ﬁlter large volumes of water

through their gills as part of their feeding activities, bio-accumulate pathogenicmicrobes,includingentericbacteria

https://doi.org/10.1016/j.bjm.2017.06.006

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andviruses.Insitustudieswithbio-accumulationofavirus indicatorinoystershaveshownthattheycaneffectively con-centrateviruses(upto99-fold)comparedtothesurrounding water.4_An_adult_blue_mussel_may_ﬁlter_∼72_L_of_water_on_an

averageperday5_and_have_been_proposed_as_bio-samplers_for

assessmentoffaecalcontaminationinrecreationalwaters.6

Consumptionofbivalvesasfoodisoftenreportedtobethe causeofdiseaseoutbreaksbyentericviruses.Thoughprimary infectionresultsfromingestionoffaecalcontaminatedfoodor water,unintentionalingestionofcontaminatedrecreational waterscanalsoleadtogastrointestinalillness.7_Norovirus

out-breakshaveadistinctseasonalitylinkedtowintermonths.8,9 NoVinfectionsarecalled“gastricﬂu”foritssimilar seasonal-ity&lackofeffectivetherapeuticslikeinﬂuenzaviruses,and alsoforitshighinfectivityandrapidevolution.10,11

Noroviruses (NoVs) are a non-enveloped positive sense

single-stranded RNA virus of the Caliciviridae family. NoVs haveasigniﬁcantimpactonhumanhealthastheyarehighly infectiousand causeacutegastroenteritis inallagegroups accountingfor>200,000 deathsworldwideeach year, espe-ciallyinchildren.12 _The_organism _is_highly_contagious_and

problematic as a hospital-acquired infection. According to WHO,<10virionsaresufﬁcienttocause infectioninadults and NoVoutbreaks oftenlead toclosure ofentirehospital wards every yearaffectingboth patientsand staff.Despite

attempts to control via ‘deep cleaning’ and implementing

hygienemeasuresoutbreakscan causeconsiderable

incon-venienceandeconomiclosses.13–15 NoVscannotbecultured oncellsanddetectionanddiagnosisincreasinglyreliessolely

on molecular methods such as reverse transcriptase-PCR

(RT-PCR).16 _Diversity _of _NoVs _both _genetically _and

anti-genically were demonstrated through RT-PCRand genomic

sequencing.17_NoVs_are_divided_into_six_genogroups_(GI-GVI)

and genogroups are further subdivided into around forty

geneticclustersorgenotypes.18_NoV_genogroups_which_infect

humansareI(NoVGI),II(NoVGII)andrarelyIV(NoVGIV).NoV GII.4isresponsiblefor80%ofthediseaseoutbreaks19_and_is

currentlythemostvirulentstraincirculatingintheUK.20_Since

ithasafastermutationratethanotherNoVs21_new_variants

emergeevery2–3yearsandisalsoresponsiblefor60–80%of outbreaksworldwide.22_The_GII.4_variant_named_Sydney₂₀₁₂

hasprogressivelyreplacedthepredecessorGII.4NewOrleans 2009variantglobally.23,24

GII.4 outbreaks occurred preferentially during winter

months25 _and _most_works _have_been_conducted_with

sam-plescollected duringwintermonths.26_Thus_the _aim_of_the

presentstudywastoscreenforNoVcontamination/retention inbivalvesduringthesummermonthsintheUKcoastal sea-waters.Thisworkcontributestothedevelopmentofaworking methodologyfortheroutinesurveillanceofmusselsroundthe yearforidentiﬁcationandgeneticcharacterizationofenteric

pathogenicvirusesaccumulatedwithinthem.

Materials

and

methods

Collectionandprocessingofsamples

Eightwildbluemussels(numbered1–8)werecollectedinApril

2013;they were attached toa nylon rope fragmenton the

metalsupportinggirdersunderMumblesPier atThe

Mum-bles, Swansea, UK. Another four mussels (numbered 9–12)

wereobtainedinJune2013fromtheRoyalDockatGrimsby, UK.Hepatopancreas, gillsand gastrointestinal tissueswere dissectedoutfromthesemussels.Tissues(∼3.0g)from mus-sels1–4,5–8and9–12constitutedthethreepooledsamples1, 2and3respectively.Musseltissuesweretriturated;digested withproteinaseKandcentrifugedat3000×gtocollect∼5mL supernatant.27_The_supernatant_was_ﬁltered_to_remove_tissue

and other debris including unwantedmicrobes(like

bacte-riaandfungi)using0.45␮Mﬁlter(Anachem-Supatop,UK)for furtherdownstreamapplications.

ViralRNAextraction,reversetranscriptionandnestedPCR Followingprocessingofthesamples,viralRNAwasextracted

usingQIAampViralRNAMinikit(Qiagen,Germany)

accord-ingtotheinstructionsofthemanufacturer.ExtractedRNA(in 60␮Lwater)wasusedforreversetranscriptiontocDNAusing BioScript RT-PCR Kit(Bioline, UK) and NoV-speciﬁc reverse primer,1422.28

ThecDNAwasampliﬁedusingNoVGI-speciﬁc(PCRNos.

443,446)andGII-speciﬁc(PCRNos.443,444)semi-nestedPCRs (ExpandHighFidelityPCRkit,Roche,Germany)accordingto previouslypublished protocols.29 _These _PCRs_target _part_of

theNoVRNApolymerase,RdRPgene.ThePCRnumberinghas

been adopted fromthe numberingofthe PCRprotocols as

previouslypublished.28

Thetargetampliconsizeswere327bpfortheﬁrstround PCR(No.443)forbothNoVGIandGII;188bpforthesecond roundPCRincaseofNoVGIand237bpforNoVGII.Positive PCR-ampliﬁedproducts(electrophoresedon1.5%agarosegel)

werepuriﬁedandcustom-sequencedforbothstrands.

The sequencing primers were the same forward and

reverseprimersoftherespectivePCRsthatresultedin visi-blebandsonthegel(e.g.primersforPCRNo.446forGINoV or444forGIINoV).

ArmouredRNA(Cells-to-cDNATM_II_Kit,_Ambion,_UK)_was

usedaspositivecontrolbyspikingextractedRNAfrom mus-selswiththearmouredRNAtotestforRTandPCRinhibitors inthemusselRNAconcentrate.

Samples1–3werefurtherscreenedforNoVGIandNoVGII capsidgenesusingnestedPCRs(PCRNos.475,476)and(PCR Nos.437,438)respectivelyandalsoforHAV(PCRNos.675,676) followingpreviouslypublishedprotocols.28

AllthreesampleswerealsoscreenedforNoVGIV(PCRNos. 603,612;546,542)andCOG4F,G4SKRandG4SKF.28,30_For_NoV

GIV detection byPCRnos. 603and 612,the reverse primer

1565usedforcDNAsynthesishadthesequenceasdescribed earlier31 _and_not_as_described _in_La_Rosa_et_al.28 _The_latter

sequenceappearstobeincorrect.Samples2and3were fur-ther testedforHEV(PCRNos.711,712)and astrovirus(PCR Nos.696,697).

Sequencing

Nucleotide (nt) sequences, conﬁrmed by bi-directional

sequencingofthePCRproducts(PCRs446,444)weresubjectto

NCBIBLASTfordetermininggeneticmatcheswithsequences

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WwithothercloselyrelatedNoVsequencesidentiﬁedfrom theBLASTsearch.Aphylogenetictreewasgeneratedbythe neighbour-joiningmethodusingMEGA632_on₁₆₉_nt_of_RdRP

gene (pertaining to nt positions 4326–4494 as in JX459908

NoVGII.4Sydneyvariant)tocharacterizetheNoVstrainsand studytheirgeneticdistanceswithothercloselyrelatedNoV strains.

Results

SequencingofNoVs

AllthreesampleswerepositiveforNoVGII-speciﬁcPCR(No. 444)(Fig.1Aand B).Samples2and 3werealsopositivefor NoV GI-speciﬁc PCR (No. 446) (Fig. 1B, sample2 isshown) andproducedPCRproductsofexpectedbandlength(188bp). AllsampleswerenegativeforGIVNoV.Samplesscreenedfor otherentericviruseswerealsonegative(withinthelimitsof

detection ofthe PCRs performed) excepta PCRproduct of

expectedsize(PCRNo.712;457bp)observedforHEVincase ofsample3.However,sequencingpooledproductfromthree suchbandsresultedinnon-HEVsequencesasalsoincaseof

non-NoVsequences observedforthe188bpbandfrom the

NoVGI-speciﬁcPCR(datanotshown).GI&GIINoVPCRs tar-getingNoVcapsidgenes(PCRNos.476,438)werenegative.

RelationshipswithotherNoVs

Phylogeneticanalysisofalignedsequencesrevealedthat

sam-ples 1and 2 from Swansea clustered closely with several

pandemicNoVGII.42012variants.Bothsamples,however,had differencesatnucleotide(nt)sequencelevels.NoVfrom sam-ple1wasnamedGII.4UK2013aandthatfromsample2was namedGII.4UK2013b.TheGII.4SydneyvariantsofNoVsfrom musselsinthepresentstudyclusteredwithNoVsofChinese andTaiwaneseorigin(Fig.1).

Thesample3fromGrimsbyclusteredcloselywithother

NoVGII.2strainsandthisNoVwasnamedGII.2UK2013(Fig.2).

NoVGI-speciﬁcPCRproductsfromsamples2and3produced

non-NoV sequenceswhich couldnot bematchedwith any

knownsequenceusingBLAST.

ThoughbothsamplescontainedNoVGII.4,the

predomi-nantstrainsaspreferentiallyampliﬁedbyPCRsweredifferent

as evident from nucleotide differences in their RdRP gene

sequences and reﬂectedin phylogenetic clustering (Fig. 2).

In the tree, prototype sequence from a NoV GII obtained

from GenBank(accession numberX86557)and anoutgroup

sequencefromaNoVGI(AF093797)wereincluded.Prototype forNoVGII.4includedinthetreewastheSydney2012 vari-ant(accessionnumberJX459907)whichinterestinglycaused highincidenceofNoVoutbreaksintheUKduring2012.Both theNoVGII.4variantsfromSwanseagroupedcloselywiththe

2012NoVGII.4humanstrains, includingthe“Sydney2012”

Fig.1–RepresentativegelelectrophoresisofsecondroundPCRproductson1.5%agarosegels,forNoVGI(PCR446)andNoV GII(PCR444)frommusselsamples1and2.(A)Musselsample1PCR.Lane1:HyperladderIV(Bioline,UK);Lane3:Negative controlforNoVGII-speciﬁcPCR(PCRNos.443,444;nuclease-freewaterastemplateforbothPCRs);ProductforPCRno.444 wasrunongel,Lane5:NoVGIIPCRproduct(237bp);Lane7:PositivecontrolforPCRinhibitorsinmussels(147bp).(B) Musselsample2PCR.Lane1:HyperladderIV;Lane2:NoVGIPCRproduct(188bp);Lane3:NoVGIIPCRproduct(237bp).

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FR669099 NoV GII Luxem 2009 FR669098 NoV GII Luxem 2009 HM635123 Hu GII 2 Korea 2009 Sample 3-Mussels 9-12 (GII.2 UK 2013) EU085490 Hu GII 2 Sweden 2004 Mussels X81879 GII 2-Msham-GBR95 GQ845370.2 Hu GII 12 StGeorge 2008 AU

JX459907 Hu GII 4 Sydney 2012 Sample 1-Mussels 1-4 (GII.4 UK 2013a)

Sample 2-Mussels 5-8 (GII.4 UK 2013b) KC688702 NV carrot salad 2012 Germany KC517362 Hu GII 4 2012 TW KF055286 Hu GII 4 Huzhou 2012 CHN KC709613 Hu GII 4 PKUPH 63 2012 CHN KC119508 Hu GII 4 2012 Spain

JQ613573 Hu GII 4 2010 AUS X86557 Hu GII 4-Lsdale-GBR-2005 X76716 NoV II 4 UK 1994

AF093797 NoV I 6 Germany 1997

75 79 85 73 92 8 58 54 19 24 95 77 67 36 54 0.5

Fig.2–PhylogeneticanalysisofthenorovirusRNA-dependentRNApolymerasegene(partial)ofseveralNoVgenogroups andgenotypes.NoVsequences,retrievedfromGenBankforcomparingwithourNoVstrainsinthemusselsareidentiﬁed bytheirGenBankaccessionnumbersontheﬁgure.Thescalebarindicatesgeneticdistanceintermsofnucleotide substitution/site/year.

variantsasdetectedearlier.24_The_most_interesting_ﬁnding_of

thisstudyisthefactthatNovGII.4Sydneyvariantswerefound eveninapilotscaleofsampleswhichadvocatesforthehigh prevalenceofthesepathogenicgenotypesinthemusselsof UKcoastalwaters.

Discussion

NoVs are most common cause ofoutbreaks of food-borne

illnesses and pose serious health risks to humans. In

the present small-scale study, mussel samples obtained

from seawater near Swansea were found to contain

two GII.4 variants. In February 2012, four wards were

closed in a nearby public hospital due to NoV outbreak

(http://www.bbc.com/news/uk-wales-south-west-wales-17110220

accessed on 14.10.2015). However,if the GII.4virus strains identiﬁed in the mussels were genetically identical to the NoVstrainsresponsiblefortheSwanseaoutbreaks,itwould demonstratethatthesevirusesprobablysurvivethesewage

treatment process and concentratein mussels. This could

notbeanalyzed inthe present study asno sequence data

ontheoutbreakofNoVstrainsfromSwanseain2012were

availableinthe public domain.In this context,it isworth mentioningthatthemostcommonlydetectedGII.4strainin thehealthcaresettingsinUKintheseason(2013–2015)was

Sydney2012(PHEMonthlyNationalNorovirusandRotavirus

Report, 14 April, 2016).33 _NoV _capsid _PCRs _were _negative

whichcouldhaveresultedfromprimermismatchascapsid

proteinsareexposedtohigherlevelofimmunepressureand

therefore, immune selection compared to more conserved

non-structural proteins like virus RNA-dependent RNA

polymerase.Consequently,thecapsidgenesaremoreprone

tohigherratesofmutation.Ashumansaremostlyinfected bythe GIIstrains, the present work indicatesthe needfor a larger study of surveillance of enteric viruses (e.g. NoV, HAVvirusandHEV,rotavirusesandothers)thatcontaminate

coastal waters of the UK and become bio-accumulated in

mussels,clamsandoysters34_conﬁrming_their_role_as_a_food

chainsourceofinfection.

NoVGIandGIIhavebeendetectedinoystersamples har-vestedfrombaysandestuariesworldwide35,36_and_also_from

humansamples.25_However,_this_is_the_ﬁrst_report,_to_the_best

ofourknowledge,onNoVGII.4Sydney2012variantdetectedin bluemusselscollectedfromUKcoastalwaters.Itsuggeststhat choosingnorovirusesasaparameterwouldbeworthyenough ifviraltestingofmusselsisintroducedonaroutinebasis.It wasalsointerestingtonotethatnorovirusishighlydetectable

evenin asmall scalesampling ofmusselsduring summer

months,suggestingthepossibleprevalenceofpathogenicNoV strainscontaminatingUKcoastalwatersthroughouttheyear. IthadbeenreportedearlierthatNoVoutbreaksaremore likelytooccurduringwinter4_as_cold_water_temperatures_and

reducedultravioletlightincreasetheirsurvival.37_The_present

workdetectedNoVstoagreatextenteveninsummermonths, soitmaybeproposedthatmusselsactasreservoirstoretain andshelterthevirusesroundtheyearsothattheycan con-tributetotheﬂuxofviableNoVscontaminationofseawaters duringwinterandpotentiategreaterspreadtocausesevere outbreaksofNoVinfectionsduringthewintermonths.

Onelimitationofthepresentworkwasthatthesamplesize wasnotlargebutstillcomparabletothatreportedearlier.28

Despitepilotlevelsamplesize,theinterestingandimportant observationwasthatdifferentpathogenicNOVGII,including

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GII.4Sydneyvariants(as evidentfrom sequence variations intheRdRpgene)couldbedetectedfrompotentiallyallthe bluemusselscollected.Thissuggestssubstantial

environmen-talcontaminationwhichperhapsexplainswhySydney2012

variantwasisolatedfrommostUKoutbreaksduring2013–15. OnepossibleexplanationfortheclusteringoftheGII.4 Syd-neyvariantsofNoVsfrommusselswithNoVsofChineseand Taiwaneseorigincouldbethatthemusselcollectionsitesin

the present study were places whereyear-round maritime

activitiestakeplace andcoastal watercontaminationfrom shipsandvesselsfromfarawayplacescouldnotberuledout. SurveillanceandcontrolofNoVoutbreaksrelatedtomussel andothershellﬁshconsumptionisthereforecrucialto pre-ventspreadofinfectioninthepopulation.Webelievethatthis workwillbeanimportantaddendumtotheongoingresearch inNoVepidemiologyintheUK.38–40

Conﬂicts

of

interest

Theauthorsdeclarethattheyhavenoconﬂictofinterest.All authorshaveagreedtotheﬁnalversionofthemanuscript.

Acknowledgements

Thefundingforthisstudywasfrom theSchoolofLife

Sci-ences,University ofLincoln and Students into WorkGrant

(awardedtoRDforRS),SocietyforAppliedMicrobiology,UK.

WethankAngelaMurtaghandDrRossWilliams,Schoolof

LifeSciences,UniversityofLincolnforcollectingthemussel samples.

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