HCMV infection disrupts barrier functions and promotes epithelial–mesenchymal transition in a cholangiocyte organoid model

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: major_revision

Abstract
Humancytomegalovirus(HCMV)isassociatedwithbileductdisordersin
immunocompetentandimmunocompromisedindividuals,includingscleros-
ingcholangitis,biliarystructures,AIDScholangiopathy,biliaryatresia(BA),
andpost-transplantbiliarycomplications.Despitetheseclinicalassociations,it
remainsunknownwhetherHCMVcandirectlyinfectthebileductepithelium
andcausepathogenesis.Here,weestablishahumaniPSC-derivedcholangio-
cyte-likecell(CLC)organoidmodelthatcanbeinfectedbyHCMV,which
resultsinreducedorganoidgrowth,adeformedstructure,andalossofbarrier
function.BulkRNAsequencing(RNA-seq)showedthattheHCMV-infected
organoidshadhighlyenrichedexpressionofgenesinvolvedinthe
epithelial–mesenchymaltransition(EMT)pathway.Importantly,blockadeof
TGF-βsignallingabrogatestheEMTinductioneffectintheHCMV-infectedCLC
organoids.Furthermore,single-cellRNA-seqindicatesthatHCMVinfection
occurredindifferentclusterswithdistinctviralandhostgeneexpression
profilesandtheanalysisshowsincreasedexpressionofEMTandTGF-βsig-
nalling-relatedgenes.ThefindingsareconfirmedinBApatientlivertissuesby
examiningHCMV-DNA+cells.TwomodesofinfectionbyHCMVarefoundin
theseCLCorganoids,whichshowproductiveandlatency-likefeatures.This
studyshowsthatHCMVinfectioninducesEMTincholangiocytesthatmay
contributetothecholangiopathyseeninvariousdiseases.

Introduction
Humancytomegalovirus(HCMV)hasbeenrecognisedasacontributor
topathologicalconditionsintheliver,particularlyinimmunocom-
promisedindividuals1.InHIV/AIDSpatientsandtransplantrecipients,
clinicaldatahasimplicatedthisvirusinsclerosingcholangitis-like
syndromes2–4 andbileductdamage5–7.However,themechanisms
remainelusive.ExperimentaldatahasshownthatHCMVcaninfect
hepatocytes,leadingtoviralreplicationanddissemination8–10.Inrat
CMV-infectedlivergraftexperiments,thepresenceoftheviruswas
showntoleadtoincreasedinflammationandseverebileductdamage
comparedwithuninfectedgrafts7.However,viralinclusionsarerarely
detectedinthebileductepitheliuminhumantransplantliver
biopsies11,12,whichleadstothequestion,doesHCMVinfectiondirectly
causecholangiopathy?
HCMVisaubiquitousDNAbetaherpesvirusthattypicallyexhibits
twomodesofinfection.Thefirstinvolvesproductiveinfectionin
humanfibroblasts,whereviralreplicationoccurs,leadingtohostcell
lysisduetotheorderedexpressioncascadeofthe~200viralgenes
encodedbythevirus.Thesecondmodeislatentinfection,which
primarilyoccursinhumanCD34+progenitorcells;inthismode,the
virusremainsdormant,withonlyasmallsubsetofviralgenesbeing
expressed13–21.Reactivationfromlatencycouldleadtodiseasesinthe
liver,whichhasbeenreportedinautoimmunehepatitis,cirrhosis,and
immunocompromisedAIDSpatients3,22–26.
HCMVcaninfectinfantsthroughcongenitalinfection,postnatal
infection(<2weeksafterbirth),perinatalinfection(3–12weeksafter
birth),oracquiredinfection(>12weeksafterbirth).Theincidenceof
congenitalHCMVinfectionrangesfrom0.4to5%27andcanresultin
varioussymptoms,includingjaundice,visionandhearingloss,sei-
zures,petechiae,anddevelopmentalmotordelay.TheHCMVinfection
rateintheperinatalperiodcouldrangefrom10to15%28;thisperiodis
importantforthedevelopmentofthebiliarytractintheliver,and
HCMV-infectedpatientsoftensufferfromliverinjury,suchaschole-
statichepatitis29,30.Inaddition,perinatallyinfectedpatientswere
foundtohaveincreasedlevelsofbileductandliverdamage-related
molecules,includingγ-glutamyltranspeptidase(γ-GT),directbilirubin
(DBIL),totalbileacid(TBA),alanineaminotransferase(ALT),and
aspartateaminotransferase(AST)31.Arecentreportshowedthat
murineCMVinfectioncouldleadtobileductdamageinmicebasedon
experimentsonCD14+biliaryepithelialcells31.Clinicaltreatmentusing
antiviralsagainstHCMV(valganciclovirorganciclovir)canimprove
serologicalbiomarkersandpatientoutcomes32,33,includingcholestasis
(e.g.,biliaryatresia(BA))ininfants34–37.
BAisararedisease(incidenceof1in5000–200,000livebirths)
thatprimarilyaffectsnewborns.Itischaracterisedbycholestasisinthe
biliarytractthatresultsfrominflammationandfibrosisoftheepithelial
cellsofthebileducts(cholangiocytes)38–40.TherearefourtypesofBA
thatareidentifiedclinically:syndromicBA(presentswithsplenic
malformation),cysticBA(presentswithobliteratedbileduct),isolated
BA(themostcommontype,presentswithinflammationandatretic
biliarytree),andHCMV-associatedBA(IgM+andassociatedwith
perinatalinfection)41.HCMVisapotentialaetiologicalfactorinthis
multifactorialdisease38,39,42,43.AlthoughHCMVisthemoststudied
humanvirusinassociationwithBA,definitiveexperimentalevidence
establishingadirectcausativerelationshipbetweenHCMVinfection
andBAdevelopmentislacking5,44,45.Clinicalstudieshavereportedthat
9.5to30.5%ofanti-HCMVIgM+BApatientshaveelevatedanti-HCMV
IgMantibodylevelsandapoorprognosis,withalowerrateofjaundice
clearance,higherincidenceofcholangitis,andmoresevereliver
fibrosisandinflammationfollowingKasai'sportoenterosomy
(KPE)12,46–48.
Recentstudieshavesuggestedthatgeneticpredisposition,
epithelial–mesenchymaltransition(EMT),andbeta-amyloidpeptide
playrolesinthedevelopmentofBA49.EMTisthebiologicalprocess
inwhichepithelialcellsundergoatransformationandstart
expressingmesenchymalcellmarkers.Thistransitionoftenresults
inthecellacquiringmoreprogenitor-likecharacteristics.Thephe-
nomenonhasbeenobservedinthebiliaryductsofpatientswith
BA50–52.TheonsetofEMTcanbetriggeredandmediatedby
inflammation,thetransforminggrowthfactorbeta(TGF-β),and
hedgehogsignallingpathways53,54.Althoughthesesignallingpath-
waysareessentialfornormaltissuemaintenanceanddevelopment,
theirexcessiveactivationcanleadtosubstantialfibrogenesis
becauseofEMT50,55,56.Moreover,inasealampreymodel,TGF-βwas
foundtobeassociatedwiththedevelopmentofBAthroughan
analysisofdifferenttranscriptionalstagesleadinguptothedevel-
opmentoftheobservedphenotype57.Inaddition,TGF-βimmuno-
histochemicalstainingwasnotedinthelivertissuesofpatients
withBA58.
Afewofthe~200genesencodedbyHCMVhavepotentialrolesin
liverandbileductdamage.Immediate-earlygene1(IE1)wasfoundto
beexpressedininfectedbiliaryepithelialcells31andcouldaffect
CYP450enzymes,alteringmetabolicpathways59.IE2regulates
SREBP1c,whichcouldpromotelipidaccumulationandleadtohepatic
steatosis60.US28,aviralchemokinehomologue,caninducecellular
signallingpathways,suchasROCK/β-cateninsignalling61.Inglio-
blastomas,US28canactivateNF-κBandtheIL-6-JAK1-STAT3signalling
axis,whichcouldinduceashifttoamesenchymalstate62,andtrigger
theRIP2/NF-κBsignallingpathway,leadingtotheupregulationofthe
expressionofmesenchymalcellmarkers(N-cadherinand
vimentin)63–65.UL144canactivateNF-κBinaTRAF6-dependentman-
ner,leadingtotheonsetofinflammation66,whileUS5-encoded
microRNAcanpromoteTGF-βtomediateEMTandfibrosis67.The
HCMVinterleukin-10(IL-10)homologuecaninducethereleaseofTGF-
βfrommonocytes,aswellastheexpressionofIE1protein,which
facilitatesglioblastomainvasion68.However,thereisalackofviralgene
expressiondataforcholangiocytesanduncertaintiessurrounding
HCMV’spreciseroleinbileductpathogenesis.
TheuncertaintiesregardingthelinkbetweenHCMVinfectionand
cholangiopathyareduetoalackofanappropriatemodeltoinvesti-
gatethedirecteffects.Toestablishsuchamodel,weusedahuman
inducedpluripotentstemcell(hiPSC)-derivedcholangiocyte-likecell
(CLC)organoidmodel69forHCMVinfection.AlthoughCLCsdonot
recapitulateallthefunctionsofcholangiocytes,theyareauteful
modelforstudyingthephenotypeofthebiliarytree,suchasdiffer-
entiationphenotypes,pathologicalresponses(e.g.,fibrosisandcho-
lestasis),andthedestructionofthebiliarystructuretomodel
cholangiopathiessuchasBA70.UsingaGFP-expressingvirus,ourdata
showsthatHCMVcaninfectcholangiocytestoproduceGFP+andGFP-
cellsub-populations.TheGFP+cellsexhibitedcharacteristicsofpro-
ductiveinfectionwithdenovosynthesisofviralparticlesfortrans-
mission.TheGFP-cellsdisplayedalatency-likemodeofinfection,with
limitedexpressionofasubsetofviralgenesaccordingtosingle-cell
RNAsequencing(scRNA-seq).Fiveclustersofcholangiocyteswere
identified,withonefullysupportingHCMVlyticinfectionandshowing
highGFPexpression.AlthoughbulkRNA-seqsuggestedtheupregu-
lationoftheEMTpathway,theinhibitionofHCMV-inducedTGF-β
signallingabrogatedtheincreasedexpressionofEMTandfibrotic
genesfollowinginfection.Inaddition,HCMVinfectiondisruptedthe
integrityandbarrierfunctionsoftheorganoids.Wewasodetected
HCMVviralDNAincholangiocytesfrom65.5%ofthelivertissues
obtainedfrompatientswithBAcomparedwith35%ofthenon-BAliver
tissues.TheHCMV-DNA+cellsexhibitedincreasedEMT-associatedN-
cadherinandTGF-βproteinexpression.Takentogether,thesefindings
demonstratethatHCMVcandirectlyinfectcholangiocyteorganoids
andinducetheTGF-β/EMTpathwayincholangiocytes.

Results
HCMVcaninfectcholangiocyteorganoids
UsingthepreviouslydescribedhiPSC-derivedcholangiocyte-likecell
(CLC)organoidmodel69,weperformeddirectHCMVinfectionusing
theTB40/E-eGFPvirusstrain,whichexpressesGFPuponlyticinfec-
tion.Byday4post-infection(PI),GFP+signalsintheCLCorganoids
exposedtoHCMVwereobserved(Fig.1a).Comparedwiththemock-
infectedcircularorganoidsthatincreasedinsizeovertime,theHCMV-
infectedorganoidsbecamedeformedandwerecharacterisedby
irregularshapesandthickenedbordersinboththeGFP+(mean±SD:
1.50±0.67μm)andGFP¯organoids(1.22±0.94μm)comparedwith
themock-infectedorganoids(0.50±0.13μm)(Fig.1a).Thenumber
andsizeoftheorganoidsintheHCMV-infectedcultureatday4PIwere
similartothoseinthemockinfectionbuttherewasasignificant
reductionatday10PI(Fig.1b,c).
TocaracterisetheHCMVinfectionoftheCLCorganoids,we
examinedtheinfectedcellsbasedonGFPsignalsandthedetectionof
HCMVproteinandDNA.Accordingtotheflowcytometryanalysis,the
frequencyofGFP+cellsrangedfrom~10to20%(Fig.1d,e);qPCR
detected~180copiesofviralDNApercell(Fig.1f).HCMVinfectionalso
occurredintheGFP-cells,wherewecoulddetect~10HCMVDNA
copies/cell,whichisapproximately20-foldlesscomparedwiththe
GFP+cellsand30-foldlesscomparedwiththeproductivelyinfected
humanfibroblasts(Hs27)(Fig.1f).Insituhybridisationusingaprobe
againstHCMVUL123detectedHCMVDNAin~28%ofcellsinthe
infectedculture,witharoundone-thirdofthemalsoshowingGFP
signals(~8%ofcells)(Fig.1g).Onlyaround0.04%ofthecellswereGFP+
andprobe¯.ToconfirmthatHCMVvirionsarebeingmadeintheGFP+
cells,immunostainingagainstpp28(ategumentprotein)wasper-
formed,whichshowedthatapproximatelyhalfoftheGFP+cellsco-
expressedpp28(Fig.1h,iandFig.S1a).Cellswithpp28signals
accountedfor~12%ofthecellsintheinfectedCLCorganoids(Fig.1h,i).
Treatmentofpermissivehumanforeskinfibroblasts(HFF-1)withthe
culturesupernatantsfromtheHCMV-infectedCLCorganoidsresulted
inGFP+fibroblasts2dayslater(Fig.1j),indicatingthepresenceof
infectiousvirions.Co-cultureofFACSortedGFP+cellsfromtheHCMV-
infectedCLCorganoidswithHFF-1cellsalsoinducedGFP+fibroblasts
byday2post-co-culture,withthevirusspreadingtonearbycells
observedondays4and7.(Fig.S1b).Incontrast,GFP+fibroblastswere
detectedonday7ofco-culturewithGFP−cells,suggestingthatreac-
tivationofHCMVDNAoccurred(Fig.S1b).Furthermore,wefoundthat
~10%oftheHCMV-infectedGFP+cellswereearlyandlateapoptotic
cells,whereasmock-andHCMV-infectedGFP¯cellsshowedsimilar
apoptosisrates,withlessthan5%oftheircellsundergoingcelldeath
(Fig.S2a,b).Importantly,RT-qPCRanalysisshowedthatHCMVgenes
(bothproductive(UL55,UL83,andUL122)andlatency-associatedgenes
(UL138,LAcmvIL10,andLUNA))wereexpressedintheGFP¯cellsinthe
infectedCLCorganoidsbuttoamuchlesserextentcomparedwiththe
GFP+cells(Fig.1k).Takentogether,thesedatademonstratedthat
HCMVinfectionofCLCorganoidscouldresultinamixedpopulation
ofcellsconsistingofthosethatsupportproductiveinfectioninthe
GFP+cells(hereafterreferredtoasHCMV-GFP+),andthosethatare
GFP¯cells,whichEithersupportlatency-likeinfectionorareunin-
fected(hereafterreferredtoasHCMV-GFP−).
BulkRNA-seqanalysisofcholangiocyteorganoidsfollowing
HCMVinfection
ToexplorehowHCMVinfectionmodulatesthephenotypeandfunc-
tionofcholangiocytes,weperformedRNA-seqonbothmock-infected
(n=5)andHCMV-infected(n=5)CLCorganoids.Principalcomponent
analysisshoweddistinctgeneexpressionprofilesbetweenthetwo
groups,withthemock-andHCMV-infectedCLCorganoidsforming
differentclusters(Fig.S3a).Heatmapanalysisdemonstrateddistinct
clustersofupregulatedanddownregulatedgenesbetweenthemock-
andHCMV-infectedorganoids(Fig.S3b).Regardingdifferentially
expressedgenes(DEGs),HCMVinfectionledtotheupregulationof
2596genesandthedownregulationof1137genescomparedwiththe
mockinfection(Fig.S3c).Usingthesedata,weexaminedifHCMV
infectioncanaltertheexpressionofgenespreviouslyreportedtobe
associatedwithBAinthesameorganoidmodel,includingAFP,APOA1,
APOA2,HNF4A,TBX3,TTR,KRT19,IL6R,SOX9,RBPJ,HNF1B,andJAG149.
HCMVinfectionupregulatedgenesinvolvedinhepatocytedevelop-
ment(AFP,APOA1,APOA2,HNF4A,TBX3,TTR,andIL6R),including
genesinvolvedintheciliaryneurotrophicfactor-mediatedand
interleukin-11signallingpathways,anddownregulatedgenesinvolved
incholangiocytedevelopment(SOX9,RBPJ,andJAG),possiblyviathe
Notchsignallingpathway(Fig.2a).Weconfirmedasignificantupre-
gulationofHNF4A,TBX3,andIL6RanddownregulationofSOX9mRNA
expressionusingRT-qPCR(Fig.S3d).CK19(acholangiocytemarker
encodedbyKRT19)andSOX9expressionlevelswerealsofoundtobe
decreasedintheHCMV-GFP¯andHCMV-GFP+organoidsintheHCMV-
infectedculturebasedonimmunofluorescenceanalysis(Fig.S4a,b).
Analysisofthefunctionalpathwaysinvolvingtheupregulatedgenes
revealedtheirstrongcorrelationwiththeEMTprocess(Fig.2b,left),
whereasthedownregulatedgenesweremostlyassociatedwiththe
androgenresponse(Fig.2b,right).
HCMVinfectionofCLCorganoidspromotesEMT
EMTisoneofthemechanismsthatcontributestothepathogenesisof
liverandbiliaryfibrosisindifferentdiseasesincludingBA50,52,71.How-
ever,theonsetofEMT,itssignificanceinBA,anditsassociationwith
HCMVarenotfullyunderstood.UsingourRNA-seqdata,geneset
enrichmentanalysisofEMTgeneswasperformed,revealingan
enrichmentscoreof0.5(Fig.S3e).ClusteringanalysisoftheEMT-
relatedgenesindicatedthatmostofthemwereconsistentlyupregu-
latedintheHCMV-infectedorganoids(n=5)comparedwiththemock-
infectedorganoids(n=5;Fig.3f).Thedifferentiallyexpressedgenes
includedCDH1,CDH2,VIM,FN1,SNAI2,TCF4,KLF8,andELF5(Fig.2c).
RT-qPCRvalidatedtheRNA-seqresultsforCDH1,CDH2,CLDN1,FN1,
SNAI2,TCF4,andKLF8(Fig.3g).Furthermore,genesinvolvedinfibrosis
(α-SMA,S100A4,HSP47,COL1A1,andCOL4A2)werefoundtobe
upregulatedafterHCMVinfectionoftheorganoids(Fig.3h).Upregu-
lationofN-cadherin(encodedbyCDH2)anddownregulationor
unchangedexpressionofE-cadherin(CDH1)areindicatorsofEMT72–74.
WesternblottingfoundthattheE-cadherinproteinlevelsofthemock-
andHCMV-infectedCLCorganoidsweresimilar,buttheN-cadherin
bandintensitywasstrongerintheHCMV-infectedorganoidscom-
paredwiththemock-infectedorganoids(Fig.2d,e).Todistinguish
betweentheexpressionofN-cadherinandE-cadherinintheHCMV-
GFP+andHCMV-GFP¯cellsintheinfectedCLCorganoids,weper-
formedimmunofluorescence(Fig.2f,g).Theresultsrevealedthatthe
HCMV-infectedCLCorganoidsexhibitedlowerexpressionof
E-cadherinintheHCMV-GFP+cellsbuttherewasnosignificantdif-
ferencebetweentheHCMV-GFP¯cellsandmock-infectedcells.
IncreasedN-cadherinfluorescenceintensitieswerefoundinboththe
HCMV-GFP+andHCMV-GFP¯cells(Fig.2f,g).Therefore,thesefindings
suggestthatHCMVinfectioncanaffectCLCorganoids,likelythrough
theinductionofEMT.
HCMVinfectionimpairsbarrierintegrityandfunctioninCLC
organoids
EMTcouldleadtothedevelopmentofthefibrosisobservedincho-
langiopathy,whichmayexplainthemorphologicalchangesobserved
intheHCMV-infectedCLCorganoids(Fig.1a).Thus,wenextsoughtto
examinewhetherthefunctionoftheCLCorganoidswasaffectedby
HCMVinfection.Wefirstperformedtheforskolin-inducedswelling
assay,whichmeasurestheactivityofcysticfibrosistransmembrane
conductanceregulator(CFTR)incholangiocytes75,76.Althoughthesize
ofthemock-infectedorganoidsincreasedby~40%followingforskolin
treatment,thesizeoftheHCMV-infectedorganoidsremainedsimilar
tothatoftheuntreatedCLCorganoids(Fig.3a,bandFig.S5a),sug-
gestingalossoftheCFTRchlorideandbicarbonateiontransport
function.BecauseEMTcanleadtothebreakdownofepithelialtight
junctions74,wetheninvestigatedwhetherHCMVinfectioncouldalso
havethiseffectasseveralstudieshavereportedthatHCMVcanleadto
thedownregulationofthetightjunction-relatedproteinZO-1on
endothelialandvariousepithelialcells77–81.IntheHCMV-infected
organoidswithGFP+orHCMV-DNA+signals,therewasadecreased
fluorescenceintensityforZO-1comparedtothemock-infected
organoids(Fig.3c,d).Wealsoperformedthetrans-epithelialelec-
tricalresistance(TEER)assay,whichmeasuresbarrierintegrity.The
HCMV-infectedorganoidshadalowervaluecomparedwiththe
mock-infectedorganoidsacrossthetime-courseexperiment(Fig.
3e).Next,thesecretorypotentialofthemock-andHCMV-infected
CLCorganoidswasevaluatedusingarhodaminetransportassay.
Thisassayusesafluorescentsubstrateforthecholangiocytesurface
glycoproteinmultidrugresistanceprotein1(MDR1)tomeasurethe
secretoryfunctionoftheorganoids.Themock-infectedorganoids
showedrhodamineaccumulationintheircellsandrhodamine
releaseintothelumen.Luminalaccumulationwaspreventedbythe
MDR1inhibitorverapamil(Fig.3f,g).TheHCMV-infectedCLCorga-
noidsshowedadiffusedstainingpattern,withrhodaminesignals
accumulatingtowardthecentre.Evenafterverapamiltreatment,the
rhodaminedyecontinuedtoaccumulateinthelumenoftheHCMV-
infectedCLCorganoids(Fig.3f,g).Inaddition,acolourimetricassay
foundthatHCMVinfectionresultedindecreasedalkalinephospha-
tase(ALP)activitylevel,whichindicatesalossofcholangiocyte
functionality(Fig.S5b).Together,thesedatashowthatHCMV
infectioninducedsubstantialchangesinthebarrierintegrityand
functionoftheCLCorganoids.
TGF-βsignallingpathwaymediatesEMTinHCMV-infected
cholangiocyteorganoids
InadditiontoitsroleinmediatingEMTincancer82–84,TGF-βis
implicatedinthedevelopmentofchronicliverdiseases85–89.Our
resultsrevealedthattheexpressionofTGFB1transcriptswasupre-
gulatedintheHCMV-infectedorganoidscomparedwiththemock-
infectedorganoidsondays5,8,and12PI(Fig.4a).TheELISAresults
alsoshowedanincreaseinTGF-βproteinreleaseuponHCMV
infectionoftheCLCorganoids(Fig.4b).Inaddition,RT-qPCRana-
lysisshowedupregulatedexpressionofTGFB1,CDH2,ACTA2,and
S100A4inboththeHCMV-GFP¯andHCMV-GFP+cellscomparedwith
themock-infectedorganoids(Fig.S6a).Furthermore,Westernblot-
tinganalysisshowedanincreasedpro-andactiveTGF-βprotein
levelsintheHCMV-GFP¯cellscomparedwiththemock-infected
organoids,whereastheHCMV-GFP+cellsappearedtobelower(Fig.
S6b–d).BlockingTGF-βsignallingusingtheinhibitorSB431542
abrogatedtheHCMV-inducedupregulationofEMT-andfibrosis-
relatedgenes(CDH2,CLDN1,SNAI2,ACTA2,S100A4,COL1A2,and
COL4A2)(Fig.4c).Strikingly,theHCMV-induceddeformityofthe
organoidsobservedonday10wasrestoredwithSB431542treat-
ment,resultinginasimilarmorphologytothatofthemock-infected
CLCorganoids(Fig.4d).ThesizeandnumberoftheseHCMV-
infectedCLCorganoidsondays4and10PIweresimilartothoseof
themock-infectedorganoids(Fig.4e–g).SB431542treatmentresul-
tedinadecreaseintheexpressionofHCMVgenes,namelyUL122,
UL44,UL83,UL55,andUL138(Fig.S7a),buthadnoeffectonthe
HCMVDNAcopynumberwithintheorganoidcells(Fig.S7b).
TreatingtheCLCorganoidculturewithablockingantibody
againstTGF-βsignificantlydecreasedtheHCMV-inducedgene
expressionofCDH2andthefibrosis-relatedgeneACTA2(which
encodesalphasmoothmuscleactin(α-SMA))comparedwithaniso-
typeantibodycontrol;theexpressionlevelsofthesegeneinthe
mock-infectedorganoidstreatedwiththeantibodiesweresimilar(Fig.
S8a).Moreover,theHCMV-GFP¯andHCMV-GFP+cellsintheinfected
CLCorganoidshadincreasedN-cadherinexpression,asmeasuredby
immunofluorescence,butthiswasinhibitedbyanti-TGF-βanti-
bodytreatment(Fig.4h,jandFig.S9).TheE-cadherinproteinlevel
appearedtobeunaffectedexceptforadecreaseintheHCMV-GFP¯
cellsinthepresenceoftheanti-TGF-βantibody(Fig.4h,iandFig.S9).
Similarly,HCMV-inducedα-SMAandS100A4expressionwasalso
hinderedbyanti-TGF-βantibodytreatment(Fig.S8b,c).Unlike
SB431542,theanti-TGF-βantibodydidnotsignificantlyalterthe
expressionofviralgenesintheHCMV-infectedCLCorganoids(Fig.
S8d),whichmaypointtoanintrinsiceffectoftheinhibitoronthe
cellularenvironmentthataffectedHCMVgeneexpression.These
findingsindicatethattheTGF-βproducedduetoHCMVinfection
couldplayaroleintheinductionofEMTinCLCorganoids.
Single-cellRNA-seqanalysisofHCMV-infectedcholangiocyte
organoids
Togainacomprehensiveunderstandingofthehostandviralgene
expressionprofilesintheCLCorganoidstoanalysetheviral–host
interactionsandinfectionheterogeneity,weperformedscRNA-seqon
themock-andHCMV-infectedCLCorganoids.Afterdatafiltering,
batcheffectcorrection,andclusteringanalysis,uniformmanifold
approximationandprojection(UMAP)wasperformed,whichrevealed
thepresenceoffiveclusters(Fig.5a).ThetopDEGsintheclustersare
showninFig.5b.KRT19expressionwashighestinClusters1,3,and4,
whereasSOX9expressionwasdetectedinClusters2–4(Fig.5b).
Furtheranalysisofthemarkersassociatedwithcholangiocytes,
hepatocytes,andprogenitorsrevealedthatCluster1represented
mature,differentiatedcholangiocyteswithmesenchymalfeatures,as
indicatedbythehighexpressionofcholangiocyte-associatedmarkers
(KRT19,KRT18,SPP1,CLDN4,andCD24)andmesenchymal-associated
markers(DCN,COL3A1,andSPARC),withthesignaturegenesbeing
COL3A1andDCN(Fig.5b,c).Thisfindingisconsistentwiththatofa
previousstudyreportingthatflat-shapedcholangiocytesexhibithigh
expressionlevelsofmesenchymalmarkers;thesecellsaretypically
foundintheliverandarederivedfromcholangiocytesundergoing
EMTandcontributetoprogressivehepaticfibrosis90.Cluster2
appearedtobealess-differentiatedcholangiocytesubpopulation
expressingslightlylowlevelsofcholangiocyte-associatedmarkersand
thelowestlevelofTACSTD291,92,withthesignaturegenesbeingCALCA
andSMOC2(Fig.5b,c).Clusters3and4exhibitedhighexpressionof
stemnessandprogenitormarkers(PROM1,CLU,TSPAN8,TM4SF4,
PPARG,ANAX4,andSOX6)(Fig.5b),whichisconsistentwiththe
findingsofpreviousstudiesonbipotentialprogenitors93,94.Cluster4
hadhigherexpressionlevelsofTFF3,APOA1,andhepatocytelineage
genes(HNF4A,ASGR1,andTTR)comparedwithCluster3(Fig.5b),
resemblinghepatocyte-basedcholangiocytes92.Amongtheclusters,
onlyCluster5exhibitedstrongexpressionofGFPandUL55(Fig.5c),
indicatingproductiveHCMVinfection(Fig.S10).Fromthedataset,
eachofthefiveclustershadonehighlyexpressedgeneperclusterthat
couldbeusedtosegregatetheclusters,namely,COL3A1,CALCA,
TSPAN8,APOA1,andPCSK1NforClusters1–5,respectively(Fig.S11).
TounderstandtheeffectofHCMVinfectionatthesingle-celllevel
intheCLCorganoids,wecomparedgeneexpressionclustersbetween
cellsfrommock-infectedorganoids,andHCMV-GFP−andHCMV-GFP+
cellsfrominfectedorganoids(Fig.5d,e).Inthemock-infectedorga-
noids,Cluster2constitutedthemajorityofcells,followedbyClusters1
and4,withCluster3havingthesmallestproportionofcells(Fig.5d,e).
Incontrast,HCMVinfectionresultedinasignificantreductioninthe
proportionofcellsinCluster2butalargeincreaseintheproportionof
cellsinCluster3.AlthoughtherewasaslightincreaseinCluster1,the
proportionofcellsinCluster4remainedsimilartothatofthemock-
infectedorganoids(Fig.5d,e),suggestingthatHCMVinfectionpro-
motestheexpressionofmesenchymalmarkersincholangiocytecells.
Cluster5exhibitedthemostobviousdifference,withHCMV-GFP+cells
dominating(Fig.5e).PseudotimeanalysisshowedthatintheHCMV-
infectedorganoids,thecellsofCluster3aswellasCluster1couldhave
arisenfromthoseofCluster2,whichwasnotfoundinthemock
infection(Fig.S12).Tobetterunderstandthecharacteristicsofthe
clusters,weturnedtogeneontologyanalysis(Fig.S13).Clusters1and2
appeartoexpressgenesinvolvedintissuedevelopmentandcilia
assemblyandorganisation,respectively.Clusters3and4express
genesthatareassociatedwithhormonesandmetabolicprocesses,
respectively.
Next,weexaminedtheviralgeneexpressionsignaturesinClus-
ters1to5.Cluster5showedhighexpressionofallkineticclasses(TC1-
7)ofHCMVgenesthatwereclassifiedpreviously20(Fig.S10),which
likelycorrespondstotheGFP+cellsintheinfectedCLCorganoidswith
replicatingHCMV.Genesthatencodeforcapsidproteins(e.g.,UL86,
UL85,UL46,andUL48A)andUL44(encodesHCMVDNApolymerase)
wereonlyhighlyexpressedinCluster5,indicatingtheproductionof
newvirionsintheHCMV-GFP+cells.Incontrast,intheHCMV-GFP¯
cells,alimitedsubsetof58viralgeneswasfoundtobeexpressedin
Clusters1to4butatlowlevels(Fig.S10andTableS1).Interestingly,33
ofthesegeneswerereportedtobeassociatedwithlatencyinCD34+
hematopoieticstemandprogenitorcells,andmonocytes(Table
S1)17,95.RT-qPCRofHCMV-GFP¯cellsfromtheCLCorganoidsexpres-
sedvariousviralgenes,althoughtheirexpressionlevelswere25-to70-
foldlowerthanthoseoftheHCMV-GFP+cells(Fig.S14).Altogether,our
experimentaldataindicatethatHCMVinfectionofCLCorganoids
resultsinnotonlyproductiveinfection(Cluster5),butalsoalatency-
likemodeofinfection(Clusters1to4).
Wenextsoughttoexamine,usingthescRNA-seqdata,whether
EMT-relatedgeneswerealsomodulatedbyHCMVinfectionintheCLC
organoidstocorroboratetheaboveexperimentalfindings.Inthe
mock-infectedCLCorganoids,theexpressionofCDH1wasmoreor
lessconsistentinClusters1–4,butintheHCMV-GFP¯cells,itwas
mostlyfoundinClusters1and3,thoughtheoverallexpressionlevel
remainedsimilar(Fig.5f).ForCDH2,therewasincreasedexpressionin
theHCMV-GFP¯cellscomparedwiththemock-infectedorganoids,
anditsexpressionwashighestinClusters1and2(Fig.5g).Interest-
ingly,forTGFB1,itsexpressionwasmainlyfoundinCluster4inthe
mock-infectedorganoids,andClusters3and4intheHCMV-GFP¯cells
(Fig.5h).TheexpressionofthesegeneswasalsofoundinCluster5.
OthergenesrelatedtoEMTweredifferentiallyexpressedinthe
infectedCLCorganoidsaccordingtothescRNA-seqdata.Whilethe
expressionofCDH1remainedsimilar,therewasanincreaseinthelevel
ofexpressionofCDH2,COL1A1,COL3A1,VIM,andS100A4intheHCMV-
GFP¯andHCMV-GFP+cellscomparedwiththemock-infectedorga-
noids,withahigherproportionofcellsexpressingthesegenes(Fig.5i).
However,therewasadecreaseinFN1expressionandalowernumber
ofcellsexpressingACTA2andMMP9.Transcriptionfactorsrelatedto
EMTwerealsoexamined,revealingincreasesintheexpressionof
SNAI1,SNAI2,TWIST2,ZEB1,andZEB2intheHCMV-infectedcells
comparedwiththemock-infectedorganoids,albeitinalowerpro-
portionofcells(Fig.5j).Furthermore,theexpressionofTGFB1,aswell
asthegenesinvolvedinthenon-canonicalTGF-βdownstreamERK
pathway(MAPK1,MAPK3,andRAF1),wasfoundtobeelevatedinboth
theHCMV-GFP−andHCMV-GFP+cells(Fig.5k).However,theexpres-
sionlevelsoftheSMAD2,SMAD3,andSMAD4genesinvolvedinthe
canonicalpathway,thereceptorgenesTGFBR1andTGFBR2,andgenes
involvedinthePI3K/Akt(PIK3CA,AKT1,andMTOR),RhoA(RHOAand
ROCK1),andTAK1pathways(MAP3K7andMAPK8)remainedsimilar
betweenthemock-infectedorganoidsandHCMV-GFP¯andHCMV-
GFP+cells(Fig.S15).Nonetheless,therewasanincreaseinthe
expressionofthetargetgenesoftheTGF-βsignallingpathway,
includingID1,ID2,CDKN1A,SERPINE1,andIL11(Fig.5k).
DetectionofHCMVDNAandincreasedTGF-β/N-cadherinlevels
inhumanBAliverbiopsies
TheabovefindingsshowthatHCMVinfectioncouldtriggerEMTina
TGF-β-dependentpathwaythatmayleadtocholangiopathiessuchas
BA.PreviousstudieshavedetectedthepresenceofHCMVinliver
biopsies,butthevariabilityhascontributedtocontroversysurround-
ingtheassociationofthisviruswiththedevelopmentofBA96.The
studiesthatusednestedorRT-PCRassaysdetectedexpressionof
HCMVgenesin0to57.1%oflivertissuesamples97–102whilethosethat
usedimmunostainingagainstHCMVproteinsdetectedthevirusin0to
55.6%ofsamples5,47,97,103.AstudyinNepaldetectedpp65in10outof13
BApatients(76.9%)inacohortofpatientswithchronichepatitis104.
However,anearlierstudythatusedinsituhybridisationfailedto
detectHCMVinthe12extrahepaticBAliversamplestheyexamined.In
ourstudy,immunohistochemicaldetectionofanHCMVantigeninBA
livertissuesfoundpositivesignalsinthebileductsandcholangiocytes
in36.3%ofthesamples(8of22)(Fig.6aandTableS2).Next,we
examinedthepresenceofHCMVDNAintheliverbiopsiesofBA
patientsusingahighlysensitiveprobetargetingHCMVUL123,com-
binedwithimmunostainingforCK19andHNF4A(ahepatocytemar-
ker).AsshowninFig.6b,HCMVDNA(white)wasdetectedinthenuclei
ofbothCK19+andHNF4A+cellsintheBAliverbiopsiesusingimmu-
nofluorescenceandconfocalmicroscopy.Tilescansrevealedless
distinctstructuresforCK19+bileductsintheBAsamplescompared
withthenon-BAsamples(Fig.6b).Thecholangiocytesin~65.5%ofthe
BAsamples(19of29)testedpositiveforHCMVDNA(TableS2).Inthe
eightsampleswherethedetectionoftheHCMVantigenyielded
negativeresults,insituhybridisationsuccessfullydetectedHCMV
DNA.ThedetectionoftheHCMVantigenorHCMVDNAinhepatocytes
inBAliverbiopsiesshowedsimilarresults.Inthenon-BAliverbiopsies
fromCCandHBcases,HCMVwaslessfrequentlydetected,withonly1
outof18(5.5%)samplesand7outof20(35%)samplestestingpositive
fortheHCMVantigenandHCMVDNA,respectively.Thepresenceof
theHCMVantigeninhepatocyteswasobservedin11of18samples
(61.1%)comparedwith9of20(45%)sampleswhenusingtheprobe
againstHCMVDNA(TableS2).Ourresultsindicatethattargeting
HCMVDNAtodetectthevirushasahighersensitivitythanusing
antibodiestodetectHCMVprotein,particularlyincholangiocytes.As
showninthetilescaninFig.7a,therewasanoverallincreaseinthe
expressionofN-cadherinandTGF-β,whichcanbeseenindifferent
regionsoftheBAlivertissue.Incomparison,therewasrelativelylow
expressionoftheseproteinsinnon-BAlivertissues(Fig.S16).Acloser
examinationrevealedthatcellswithstrongHCMV-DNA+signalsinthe
nucleihadmoreprominentexpressionofbothN-cadherinandTGF-β
comparedwiththosethatareHCMV-DNA¯intheBAlivertissues,as
evidencedbytheexpressionofalimitedsubsetofviralgenes,
includinglatency-associatedgenes,alowviralDNAcopynumber,a
lackofinfectiousvirusproduction,andevidenceofreactivationafter
prolongedco-culturewithfibroblasts.WhileHCMV-infectedlyticcells
willlikelyrapidlyundergocelldeath,thepersistenceoflatency-like
infectedcellscouldbevitalforthesustainedandprogressivebileduct
pathologyobservedinliverdiseasessuchasBA.Therolesofthe
detectedviralgenesincholangiocytesthatmaymediatetheEMTand
theTGF-βpathwayshouldbestudied(TableS3)todetermineifthisis
themechanismthroughwhichHCMVcontributestothedevelopment
orprogressionofBAandtoexploretheirpotentialapplicationas
biomarkersforthediagnosisofHCMV-relatedBA.
Intrahepaticlargeandsmallcholangiocyteslinethesmalland
largebileducts,respectively,intheafferentdirectionfromtheliver,
andtheyexhibitdistincttranscriptionalcharacteristics122–124.Thesetwo
typesofcholangiocyteshavedistinctfunctions:smallcholangiocytes
canrespondtocarbontetrachloride-inducedbileductdamageandare
consideredproliferativeormigratory,whereaslargecholangiocytes
canrespondtosecretinandsomatostatin,primarilyhaveproliferative
functionsduringbileductligation,anddisplayinflammatory
activity125–129.Studieshavebeenconductedusingcholangiocytesfrom
rodents,buttherearenostudiesexaminingHCMVinfectionofsmall
orlargecholangiocytesortheirsubtypes.OurscRNA-seqdata
demonstratedthepresenceofHCMVviralgeneexpressioninallfive
identifiedclusters,withCluster5exhibitingthehighestlevelsofviral
getranscripts,whichcorrelatedwithhighGFPexpression.This
findingindicatestheoccurrenceofa‘full-blown’productiveinfection.
RecentscRNA-seqstudiesofthehumanlivertranscriptomehave
identifieddifferentclustersofcholangiocytes,hepatocytes,and
progenitor-likecells92,124,130.Theclustersidentifiedinourcholangio-
cyteorganoidmodelhavesertainsimilaritiestothosereportedby
Andrewsetal.(2022)92.Intheirstudy,theyidentifiedsixclustersof
cholangiocyte-associatedcellsfromhumanliversamples.Oneoftheir
clusters(Chol-3)thathighlyexpressedhepatocytemarkersalso
showedsomedegreeofexpressionofprogenitor-associatedmarkers;
thisissimilartoClusters3and4inourdata.However,ourCluster
1showedmoreexclusiveexpressionofcholangiocytemarkersand
lackedexpressionofprogenitor-associatedorhepatocytemarkers,in
contrasttoChol-4intheirstudy.Thesefindingsindicatethepresence
ofintrinsicdifferencesbetweentheclustersidentifiedfromliver
samplesandthosederivedfromiPSC-derivedcholangiocyteorga-
noids.RegardingtheroleofHCMVinBA,ourfindingsrevealedthatthe
viruscaninfectcholangiocyteswithdifferenttranscriptionalpheno-
types.HCMVinfectionorexpressionofitsgenesmightalterthe
transcriptomesofthedifferentclusters,resultingindifferenteffects.
Inourdata,Clusters3and4intheHCMV-infectedCLCorganoids
showedthehighestexpressionofTGFB1,whichmaymediatethe
inductionofCDH2intheotherclusters,namely,1and3.Thisfinding
suggeststhatintheliverandbileduct,therecouldbeasubtypeofcells
thatsupportHCMVlatency-likeinfectionandexpressTGF-βtoacton
bystanderhepatocytesorcholangiocytestoinduceEMT.Itwouldbe
interestingtodefinethedifferenttypesofcholangiocytesinliver
biopsiesbasedonthesignaturephenotypicmarkersidentifiedabove
andtoperformspatialomicsstudiestofurtherdefinetheinteractions
betweenthedifferentcellsintheliver131.
EMThasrecentlybeenshowntobeanimportantfactorinthe
developmentofBA.OurdatashowedthatinHCMV-infectedCLC
organoids,N-cadherinexpressionisupregulatedandEMT-related
transcriptionfactorsaremodulated,whichmayhavecausedthelossof
barrierandorganoidfunctions.EMTcanbeinducedthroughthe
activationofthesonichedgehog(SHH)orTGF-βsignalling
pathway53–55,71,aswellasinnateimmuneresponsesthroughtheDNA
sensorscGASandIFI1650,132–135.EMTpromotesthemesenchymalpro-
genitorphenotypeandfibrogenesisincholangiocytesininfantswith
BA71.Moreover,HCMV,EBV,andhepatitisBandCvirusescanupre-
gulateSHHandtheinnateimmunepathwaystomediateEMT136–138,but
nospecificdataareavailableonEMTinBA.RT-qPCRshowedthat
HCMVinfectionincreasedthegeneexpressionofproinflammatory
cytokines(TNF-αandIL-1β)inCLCorganoids(Fig.S17a),andscRNA-
seqanalysisshowedincreasedgeneexpressionofTNFA,IL1B,IL18,
CXCL8,NFKB1,NFKB2,IKBKB,andCHUK(Fig.S17b).Itwasexpected
thatthesegenes,aswellascGASandIFI16,wouldbeupregulatedinthe
HCMV-GFP+cellsduetoviralreplicationandtheabundanceof
pathogen-associatedmolecularpatterns(PAMPs);however,itshould
benotedthattheywerealsoelevatedtoalesserextentintheHCMV-
GFP¯cells.Ontheotherhand,afewstudieshavedemonstratedthat
HCMVinfectioncanmodulatetheSHHpathway139,140.OurscRNA-seq
dataindicatedthatSMO,SHH,andSUFUwereupregulatedinthe
HCMV-GFP¯cells,whereasKIF7,PTCH2,GLI1,andGLI2wereupregu-
latedintheHCMV-GFP+cells(Fig.S17c).RegardingtheTGF-βsignalling
pathway,UL22Aexpressionwasdetected,suggestingthatthe
expressionoftheencodedmiRNAsCansuppresscanonicalSmad2/
3signalling67,whichcouldexplainwhytheSMAD2,SMAD3,andSMAD4
geneexpressionlevelsinthemock-andHCMV-infectedCLCorganoids
weresimilar,whereasgenesinvolvedinthenon-canonicalSmad-
independentERKpathway,aswellasdownstreamtargetgenes,
appearedtohaveincreasedexpressionafterHCMVinfection.This
arrangementcouldbeadvantageousforHCMVpersistenceandto
preventtheonsetoftypeIinterferon(IFN)responses67,141.Itwasalso
interestingtoobservethatSB431542,butnottheanti-TGF-βblocking
antibody,coulddownregulateHCMVgeneexpressionintheinfected
CLCorganoids,whichsuggeststhatthedownstreamsignallingpath-
waysmayplayarole.SB431542canalsoinhibittheactivinpathway,
whichiscloselyassociatedwithTGF-βsignalling.Otherresearchers
haveshownthatSB431542ortheinhibitionofactivincouldreduce
SARS-CoV-2geneexpressionandreplication142,143.WhetherHCMVis
affectedbysimilarmechanismsremainstobeinvestigated.HCMVcan
induceTGF-βsignallinginlatentlyinfectedCD34+progenitorsandin
productivelyinfectedepithelialcells67,144.TGF-βsignallinglikelyacti-
vatedEMTandfibroticgenestoresultintheobserveddetrimental
effectsontheorganoidintegrityandcell-barrierfunction.Future
studieshouldexplorewhetherHCMV-positivepatientswithBA
exhibitincreasedTGF-βsignallingintheirbileductanddeterminethe
additionalrolesofthiscytokineinliverfibrosisandrelateddiseases145.
DemonstratingthatHCMVinfectioncanoccurincholangiocytes
representsasubstantialadvancementinourunderstandingofHCMV-
associatedcholangiopathyandopensupthepossibilityofexploring
whetheranti-HCMVimmuneresponsescontributetotheprogression
ofliverdiseases,includingBA.Tcellsisolatedfrom56%ofBAliver
samplesrespondedtotheHCMVpp65antigenorHCMVprotein
lysatesbyproducingIFN-γ.Inaddition,therewasanincreaseinanti-
HCMVIgMantibodylevelsandareductioninperipheralbloodreg-
ulatoryTcells(Tregs)146.However,laterstudiesdidnotfindsignificant
differencesinTregpopulationsbetweenpatientswithandwithout
BA147,148.SomeBApatientsexhibitedaninfiltrationofTh17cellsintothe
liver,whichwascorrelatedwithpoorsurgicaloutcomesfollowing
KPE149.ThesefindingssuggestthatinflammatoryT-cellresponses
mightplayaroleinmediatingdamagetotheliverandbiliarysystemin
BA.HCMV-specificTcellsmightmediateimmuneresponsesagainst
HCMV-infectedcholangiocytespost-KPE,leadingtounfavourable
clinicaloutcomes.OurHCMVinfectionorganoidmodelcanbeusedin
co-cultureexperimentswithautologousHCMV-specificTcellsto
determinewhethertheseTcellscanpromoteinflammationand
fibrosisresponsesinorganoids.Moreover,thestrengthoftheT-cell
responsecanbemeasurecanbemeasuredtodetermineifthepresentationofHCMV
antigensinducesthe-releaseoftheTh1cytokinesIFN-γand
osteopontin150–152ormediatesCD8+T-cellcytotoxicityagainst
cholangiocytes153.Thesestudiescouldprovideinsightsintothelong-
termeffectsofHCMVinfectionandhowanti-HCMVimmunerespon-
sessustainchronicinflammationinBAlivers.
AlthoughourfindingsdemonstratedthatHCMVcaninfectcho-
langiocytesinanorganoidmodel,somefundamentalquestions
remainandneedtobeansweredbeforeestablishingadirectlink
betweenHCMVandclinicalBA.CongenitalHCMVinfectionrarely
resultsinliverdiseases,includingBA.EvenifHCMVinfectiondevelops
duringinfancyorlaterinlife,orinlivertransplantrecipientswith
HCMVviremia,itdoesnottypicallydevelopintocholangiopathy.
Thus,thereexistsaknowledgegaptobebridgedtounderstandthe
roleofHCMVinthedevelopmentofBAoritsimportanceundercertain
geneticpredispositionscenarios.We,andothers,havefoundthatBA
patientsexhibitgeneticmutationsingenessuchasADD3(whichplays
aroleinprimarycilia),KIF3B(playsaroleinciliaryfunctionofcho-
langiocytes),MAT1A(playsaroleinmethioninemetabolism),andTHB1
(playsaroleinextracellularmatrix)38,154–158.WhetherHCMVexploitsthe
geneticenvironmentinsomeBApatientstoestablishpathogenic
effectsincholangiocytesremainstobeexplored.

Methods
Virus
TheTB40/E-eGFPviruswasgeneratedbytransfectingabacterialarti-
ficialchromosome(BAC)containingthecompletesequenceofhuman
herpesvirus5strainTB40/E(TB40-BAC4clone),whichwasmodifiedto
consistofaneGFPreporterunderthecontroloftheSV40promoter
(TB40/Ewt-GFP)(kindlyprovidedbyEainA.Murphy)21.Originally,the
TB40-BAC4wasderivedfromcloningtheendotheliotropicTB40/E
isolateofHCMVintoaBAC159,160.Togeneratevirusstocks,low-passage
humanforeskinfibroblast1(HFF-1)cellsweretransfectedwiththe
pCGNpp71plasmidwiththeBACDNA.Viruseswereharvestedfrom
thetransfectedculturebycollectingthecellsandmedia,whichwere
centrifugedat3100×gfor30min.Thesupernatantwasusedtoinfect
HFF-1cells.Thecellsweremonitoreduntilthemaximumcytopathic
effectwasobserved(at10–14days)beforecollectingthemedia,which
wascentrifuged,titrated,andusedatamultiplicityofinfection(MOI)
of0.01toinfectHFF-1forviralpropagation.Thevirusstockswere
storedinDulbecco’sminimalessentialmedium(DMEM)supple-
mentedwith5%foetalbovineserum(FBS)(Cat.No.10270106;GIBCO)
at–80°C.
Cellsandorganoids
hiPSCswerederivedfromtheperipheralbloodofhealthyindividuals
andweresubjectedtoepisomalreprogrammingusinganepisomal
plasmidexpressingOCT3/4,shp53,SOX2,KLF4,L-MYC,andLIN28
(plasmidspCXLE-Hoct3/4-shp53,pCXLE-Hsk,andpCXLE-Hul;
Addgene).TheuseofhumancellstogenerateiPSCswasapprovedby
theInstitutionalReviewBoardoftheUniversityofHongKong/Hospital
AuthorityHongKongWestCluster(HKU/HAHKWIRB;reference
numberUW22-127).Informedconsentwasobtainedfromallhuman
researchparticipants.ThehiPSCswereculturedinStemFlexmedium
(Cat.No.A3349401;LifeTechnologies)at37°CinGeltrex-coatedsix-
wellplatesandpassagedevery3daysusingStemProaccutase(Cat.No.
A11105-01;GIBCO).Themediumwaschangeddaily.
ThehiPSCsweredifferentiatedintocholangiocyte-likecell(CLC)
organoidsusingapreviouslydescribedprotocol69.Briefly,thediffer-
entiationprocessinvolvedfivemainstages:definitiveendoderm(DE)
(days1–3),foregutprogenitor(FP)(days4–8),hepatoblast(HB)(days
9–12),andcholangiocyteprogenitor(CP)(days13–16).Inthefinal
stage,theCPswereplacedinorganoidculturemedium(TableS4)and
werebrokenintosmallclumpsandseededintoMatrigel(Cat.No.
35623;Corning)ina24-wellplatetofacilitatethereformationof3D
organoids.
HFF-1(#SCRC-1041)andHs27(#CRL-1634)cellsarebothhuman
foreskinfibroblastsandwerepurchasedfromATCC.Thecellswere
maintainedinDulbecco’sminimalessentialmedium(DMEM)supple-
mentedwith15%foetalbovineserum(FBS)(GIBCO)andculturedat
37°Cwith5%CO2.HFF-1cellswereusedforHCMVviralpropagation
andfortestingviraltransmissionfromCLCorganoidsupernatantsor
co-culturedwithinfectedcells(ataratioof1.5:1organoidcells/HFF-
1cells)fromCLCorganoids.Hs27cellswereusedforproductive
infectiontomeasurethenumberofHCMVDNAcopiesinthecells
usingqPCR,whichisdescribedbelow.
HCMVinfectionofCLCorganoids
After10daysofdifferentiation,theCLCorganoidsweredissociated
intoinglecellsusingTrypLE(Cat.No.12604-013;ThermoFisher
Scientific)andwashedwithAdvancedDMEM/F12medium(Cat.No.
12634028;ThermoFisherScientific).FortheHCMVinfection,the
dissociatedcellswereplacedinDMEM/F12mediumcontaining
HCMVatanMOIof1ina15mlcentrifugetube,whichwasthen
placedsidewaysinacellcultureincubatorfor3hat37°Cwith5%
CO2.Thetubewasgentlytappedevery1htoshufflethecellsand
viralinoculum.Themockinfectionwasperformedinparallelusing
theequivalentvolumeofmediaastheinoculum.Afterincubation,
themock-andHCMV-infectedcellswerewashedtwotimeswithPBS,
resuspendedinMatrigel,andseededintoa24-wellplatetoform3D
organoids.
Totestwhetherinfectiousvirionswerereleased,undilutedcul-
turesupernatantfrommock-andHCMV-infectedCLCorganoidswere
usedtocultureapermissiveHFF-1monolayerat70-80%confluence.A
totalof2.2×104cellsperwellwereseededovernightina24-wellplate.A
mock-andHCMV-infectedcellswereassociatedfromtheCLCorga-
noids,washed,FACSortedintoGFP¯andGFP+cells,andaddedtothe
HFF-1monolayerataratioof1.5:1.TheGFPsignalsweremonitored
overtimeusinganinvertedfluorescencemicroscope(NikonEclipse
Ts2R)at100×magnification.
Determinationofthesizeandnumberoforganoids
Stereoscopicimagesofthemock-andHCMV-infectedorganoidswere
capturedusingtheIncucyteS3Live-CellAnalysissystem(Sartorius);
thesoftwaretoolinthissystemcanbeusedtoanalysethesizeand
numberoforganoids.High-resolutionimageswerecapturedusinga
NikonEclipseTs2Rinvertedmicroscopeat100×magnificationto
evaluatethemorphologyandGFPfluorescenceintensityofthemock-
andHCMV-infectedorganoids.
Immunofluorescencestaining
Thestainingprocedureswereperformedaccordingtoapublished
protocol161.Theorganoidswerefixedusing4%paraformaldehyde,
permeabilisedby0.1%TritonX-100,andblockedusing10%donkey
serum.Thiswasfollowedbyincubationwithprimaryantibodiesfor
pp28(1:100,Cat.No.ab6502;Abcam),E-cadherin(1:200,Cat.No.
14472S;1:1000,Cat.No.3195S;CellSignalingTechnology),N-cadherin
(1:700,Cat.No.13116;CellSignalingTechnology),S100A4(1:250,Cat.
No.ab197896;CellSignalingTechnology),α-SMA(1:200,Cat.No.
A5228;Sigma-Aldrich),TGF-β(1:50,Cat.No.sc-130348;SantaCruz
Biotechnology),andZO-1(1:50,Cat.No.402200;Invitrogen)at4°C
overnight,andthenincubationwithsecondaryantibodiesatroom
temperaturefor1h.ImageswereacquiredusingaLeicaStellariscon-
focalmicroscopeandanalysedusingLeicaLASXsoftwareandImageJ
software(NIHversion1.47).
Westernblotting
Onday11PI,theCLCorganoidswereextractedusingCellRecovery
Solution(Cat.No.354253;Corning)fromMatrigelandlysedwithlysis
buffer.Thesamplewasboiledfor10minat70°Candsubjectedto
sodiumdodecyl-sulphatepolyacrylamidegelelectrophoresis(SDS-
PAGE).Then,theproteinsweretransferredtoaPVDFmembraneand
blockedin5%bovineserumalbumin(BSA;ThermoFisher)inTBS-Tfor
1hatroomtemperature.Themembranewasincubatedwiththepri-
maryantibodyovernightat4°C.Theprimaryantibodiesusedwere
antibodiesforE-cadherin(1:200,Cat.No.14472S;CellSignaling
Technology),N-cadherin(1:700,Cat.No.13116;CellSignalingTech-
nology),TGF-β(1:400,Cat.No.ab215715;Abcam),andβ-actin(1:1000;
Cat.No.MABT523;MerckMillipore).Thenextday,themembranewas
incubatedwiththecorrespondingsecondaryantibodyfor1hatroom
temperatureandvisualisedwiththeSuperSignal™WestPicoPLUS
ChemiluminescentSubstrate(Cat.No.34579;ThermoFisherScientific)
andChemiDoc™MPImagingSystem(Bio-Rad).
FlowcytometryanalysisofcellsintheCLCorganoids
Themock-andHCMV-infectedCLCorganoidsweredissociatedinto
singlecells,analysedforGFPsignals,andthenFACSortedintoGFP¯
andGFP+sub-populationsusingaBDFACSMelodyTMinstrument(BD
Biosciences).ThesortedcellsweresubsequentlyusedforRT-qPCR
andWesternblotting.Toexaminecellviability,thecellsfromdis-
sociatedCLCorganoidswerestainedusingtheAPCAnnexinVApop-
tosisDetectionKitwithPI(Cat.No.640932;BioLegend)accordingto
themanufacturer’sinstructions.DatafortheAnnexinVandpropidium
iodidestainingwasacquiredusingaBDFACSymphonyTMA1flowcyt-
ometer.AnalysisofthedatawasperformedusingFlowJosoftware
v.10.8.1(FlowJo,LLC).
DetectionofHCMVDNAandTGF-βandN-cadherinlevelsinBA
andnon-BAlivertissues
TheuseofhumanpatientlivertissueswasapprovedbytheInstitu-
tionalReviewBoardoftheUniversityofHongKong/HospitalAuthority
HongKongWestCluster(HKU/HAHKWIRB;referencenumbersUW
20-575andUW16-052).Informedconsentwasobtainedfromall
humanresearchparticipants.Paraffin-embeddedlivertissuesections
frompatientswithBAandnon-BA(e.g.,withacholedochalcyst(CC)or
hepatoblastoma(HB))wereincubatedwithprimaryantibodiesagainst
CK19(Cat.No.ab220193;Abcam),HNF4A(1:2000,Cat.No.ab201460;
Abcam),N-cadherin(1:400,Cat.No.CST-13116;CellSignalingTech-
nology),andTGF-β(1ː50,Cat.No.sc-130348;SantaCruz)overnightat
4°C.Post-primaryantibodyfixationwasperformedusing10%neutral
bufferedformalinfor30minatRT,followedbyaproteasetreatment
for30minat40°C.Next,insituhybridisation(ISH)forHCMVDNAwas
performedwithafluorescenceprobeagainstHCMVUL123(Cat.No.
502271;ACDBio)followingtheinstructionsintheRNAscopeMultiplex
FluorescentReagentkitV2(Cat.No.323100;ACDBio).Then,thetissue
sectionswereincubatedwithfluorophore-conjugatedsecondary
antibodiesfor30minatRTandcounterstainedwith4′,6-diamidino-2-
phenylindole(DAPI).ImageswerecapturedusingaLeicaStellaris
confocalmicroscope.Fortheimmunohistochemistryanalysisof
HCMVproteins,theprimaryantibodyusedwasanti-CMV(1:50,Cat.
No.IHC560)fromCreativeBiolabs.DAB(Cat.No.K3468;Dako)was
usedfordevelopment.ImageswereacquiredusingaNikonEclipse
E600microscope.
Reversetranscriptionandquantitativereal-timePCR
TotalRNAwasextractedusingRNAisoPlus(Cat.No.9109;Takara
Bio)andreverse-transcribedintocDNAusingthePrimeScriptRT
ReagentKitwithgDNAEraser(Cat.No.RR047Q;TakaraBio)in
accordancewiththemanufacturer’sinstructions.QuantitativePCR
(qPCR)wasperformedusingaStepOnePlus™Real-TimePCRSystem
andtheTBGreen®PremixExTaq™II(TliRnaseHPlus)kit(Cat.No.
RR820A;TakaraBio).Therelativeexpressionlevelsofthetargetgenes
inthemock-andHCMV-infectedorganoidsweredeterminedusingthe
2−ΔΔCTmethod.
TheqPCRanalysisofHCMVDNAwasperformedasdescribed
previously162.Briefly,HCMVDNAwasextractedfromcellsusing
NucleoSpinforDNAkits(Cat.No.740952;Macherey-Nagel).Quanti-
ficationoftheHCMVgenomecopynumberwasperformedbytar-
getingtheHCMVUL55gene,whilethehumanCCR5genewasusedasa
referenceforestimatingthenumberofcells162.Alltheprimersusedare
listedinTableS5(ThermoFisherScientific).
Rhodaminetransportassay
Theorganoidswereincubatedinorganoidculturemediumwithor
withoutverapamil(10µM;Sigma-Aldrich)at37°Cfor60min.Next,the
organoidswereincubatedwith100µMrhodamine6Gdye(Invitrogen)
inorganoidculturemediumat37°Cfor10minandthenwashedthree
timeswithDPBS(Cat.No.14190144;GIBCO).Theorganoidswere
imagedusingaNikonEclipseTs2Rinvertedmicroscope.
Forskolin-inducedswellingassay
Theforskolin-inducedswellingassaywasperformedtoexaminethe
cysticfibrosistransmembraneconductanceregulator(CFTR)activity
intheorganoids.Thisassaywasperformedfollowingapreviously
publishedprotocolwithminormodifications163.Briefly,theorganoids
wereculturedfor5daysPIandimaged0,3,6,and24hafterthe
additionofforskolin(10µM;Cat.No.1099/10;TocrisBioscience)and
3-isobutyl-1-methylxanthine(IBMX,100µM;Sigma-Aldrich)usingthe
IncucyteS3Live-CellAnalysisSystem(Sartorius)at37°C.Toquantify
thepercentageofswelling,areasoftheorganoidsweremeasured
usingImageJ.
CLCorganoidcellmonolayercultureandtrans-epithelialelec-
tricalresistance(TEER)measurement
Afterinfection,theformedCLCorganoidswereexpandedforone
passageandculturedfor8daysbeforebeingextractedfromthe
MatrigelandtreatedwithTrypLEfor5minat37°Ctoobtainsingle
cells.Thecellsuspensionsweredilutedto1×106cell/mLwithorganoid
mediumcontaining10μMY-27632(Cat.No.1254/1;TocrisBioscience).
A100μLvolumeofthecellsuspensionwasseededintotheapical
compartmentofa24-welltranswellcellcultureinsertand600μLof
organoidmediumwasaddedtothebasalcompartment.Theculture
mediumintheapicalandbasalcompartmentasremovedand
replacedwith200and500μLofDPBS,respectively,beforemeasuring
thetrans-epithelialelectricalresistance(TEER)usingtheEVOMTM
ManualMeter(WorldPrecisionInstruments).Theresistance(R)across
thecellmonolayerwasmeasuredinohms(Ω).Thecorrectedresis-
tanceacrossthecellmonolayerwasobtainedbysubtractingthevalues
ofthewell-onlycontrolcontainingDPBS.TheTEERvalueswerenor-
malisedtotheunitarea(0.33cm2poresize):Ω×0.33cm2.
Bulkandsingle-cellRNAsequencing
Singlemock-andHCMV-infectedcholangiocyteorganoidsatday10PI
wereretrievedfromtheMatrigelandplacedintoindividualtubesand
processedusingtheSmart-seq2.0protocolwithminormodifications
togeneratebulkRNAtranscriptomes164.Forsingle-cellRNAsequen-
cing(scRNA-seq),cellcountingwasperformedusinganInvitrogen
CountessIIFLautomatedcellcounter,andanapproximately1×106
cells/mLcellsuspensionwith>80%viabilitywaspreparedandsentto
10×GenomicsforanalysisusingtheCellRangerv10.0(10×Genomics)
analysispipeline.Thegeneexpressionmatriceswerefiltered,nor-
malised,andclusteredusingtheSeuratv5Rpackage.Cellsthat
expressedfewerthan500ormorethan9000genes,hadagreater
than20%mitochondrialgenecontent,orhadfewerthan1500ormore
than80,000uniquemolecularidentifiers(UMIs)wereremoved.
Pseudotimeanalysisofthetrajectoryofdifferentclusterswasper-
formedinRusingMonocle3package.RStudioversion4.5.0wasused
fordatavisualisationandstatisticalanalysis.
Statisticalanalysis
AllstatisticalanalyseswereperformedusingGraphPadPrism8.0.The
statisticalmethodsusedfortheexperimentsareindicatedinthefigure
legends.Alldatawerepresentedasthemean±standarddeviation.
Analysisofvariance(ANOVA)orStudent’st-testwasusedtocompare
twoormoregroups.Differencesbetweenexperimentalgroupswere
consideredsignificantifP<0.05.
Reportingsummary
FurtherinformationonresearchdesignisavailableintheNature
PortfolioReportingSummarylinkedtothisarticle.
Dataavailability
ThebulkRNA-seqandscRNA-seqdatageneratedinthisstudyhave
beendepositedintheNCBISequenceReadArchive(SRA)website
(https://www.ncbi.nlm.nih.gov/sra/?term=)databaseunderaccession
codesPRJNA1181225andPRJNA1181820,respectively.Theexperi-
mentaldatageneratedinthisstudyareprovidedintheSupplementary
Information/SourceDatafile.Sourcedataareprovidedwiththispaper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Zuodong Ye et al presents an iPSC derived cholangiocyte model system to study mechanisms involved in Biliary Atresia and specifically the role of CMV infection in accentuating this process is modeled and investigated. Using this modeling combined with bulk and single cell RNA sequencing approaches and other techniques the authors determine a mechanistic link between CMV infection and Biliary Atresia via TGF-B EMT and disruption of barrier function. This is an interesting paper using a relevant model to examine the underlying mechanisms for this disease. My main concerns listed below have to do with the quality and lack of quantitation of some of the data presented (such as infection, IF and western blots) and the consequent conclusions drawn from the data, which need to be strengthened and addressed.\n\nThe manuscript does not adequately describe how cholangiocyte organoids were infected. For example, it is unclear if spin infection was used or if the mock infection protocol was identical. How long was the infection? Was it in suspension, in a pellet or in a plate? These details should be included for reproducibility.\n\nIt is implied that the model supports HCMV replication and viral production - referring to the single cell RNA sequencing data, the manuscript frequently mentions that the model demonstrates \"cells with high levels of viral replication\" (line 347) or \"producing new viruses for transmission.\" However, this claim overstates the evidence presented. GFP expression alone is not sufficient to confirm the production of infectious virions necessary for transmission. To definitively demonstrate that cholangiocytes support productive HCMV infection, the authors would need to use the supernatant from infected organoids, which should contain infectious virions if replication is complete. This supernatant could then be used to infect other organoid susceptible cell line models to confirm productive infection.\n\nWhat is the percentage of infected organoids or percentage of infected cholangiocytes per all cells that are obtained with this model? From Fig 1F and 1G, the infection efficacy does not seem to be very high. The authors should dissociate the organoids to single cell level and quantify infection rates together with other relevant markers by Flow cytometry.\n\nLine 124 – 125 Figure 1 A states that the morphologies were different and organoids had a thicker boundary. This is not very clear in the representative image. Can the average length of the boundary be measured and quantified with numbers?\n\nFigure 1D: Why are the copies per cell decreasing over time? In addition, the control shows copies in fibroblast cell after 48 hours. This should also be quantified at a later timepoint to see if the viral counts go down because of lysis of infected cells. This could also serve as a reason for decrease in copies in the organoid model. If not, then viability of organoid needs to be measured with time. This graph also needs an appropriate negative control such as a heat-inactivated virus.\n\nFigure 1F and G is not very convincing by the image provided. It is not apparent that the signal for CK19 and SOX9 are lower in infected organoids. This needs to be quantified by measuring the intensity of the IF signal.\n\nFigure 2 and 3 represent data from the bulk RNA sequencing analysis comparing infected and uninfected cholangiocyte organoids and should be merged into one figure. Figure 2D and 3C should be presented in supplementary. Figure 2B and 3B can be merged. Figure 2E, 3D and 3E are validation of DEGs from the RNA-seq experiment. They can all be merged into one supplementary figure.\n\nFigure 2D and 3C: Is this data from the RNA seq? Or validated in the same organoid models and donors? If so, the number (N) needs to be indicated in figure legends and individual points need to be indicated on the graph.\n\nFigure 3F: This is the only functional readout of EMT transition presented, and similar to previous comment on Figure 1F conclusions cannot only be made based on one representative microscopic image. The intensity of the signal needs to be quantified and other functional readouts of EMT transition at protein level needs to be measured. Using flow cytometry strategy as discussed above, the authors should examine the expression levels of E and N cadherin by staining with antibodies and measuring differential expression at the protein level in the GFP positive (infected) and GFP negative (uninfected) cholangiocytes within their organoids.\n\nFigure 4, Viability of organoids needs to be measured after infection. From the representative images it appears that organoid viability is being severely compromised and this could explain the loss of barrier function.\n\nThe authors should also examine cholangiocyte functionality such as via alkaline phosphatase activity in presence and absence of HCMV infection both at early and later timepoints, to gain more insight into the effects of HCMV infection on cholangiocytes.\n\nFigure 5: In general the authors need to quantify IF images and western blots (how many times was this performed?), particularly those measuring N and E cadherin since this is one of the main markers for EMT transition investigated.\n\nFigure 5A: Did the authors also see TGF-B expression upregulated in their RNA-seq dataset (that was at 10 dPI). If not, do the authors have an explanation about why this was not observed in their RNA-seq.\n\nFigure 5B: The western blot needs to be quantified from at least 3 separate blots from 3 independent experiments with different donors to make a statement that N and E cadherin levels are being up or down regulated.\n\nLine 252: Why is SB causing decrease in expression of CMV genes? Is the reversal of EMT phenotype actually controlled by TGF-B signalling or by the CMV genes? Is the inhibition of TGF-B signalling also causing inhibition of CMV genes? If so, are the CMV genes resulting in EMT transition, not necessarily via a TGF-B reversal pathway?\n\nHow do CMV induced genes dysregulate TGFB signalling and why inhibiting TGF-B inhibit the CMV gene expression, which is the chicken and which the egg. The authors should speculate/discuss.\n\nFigure 5: SB effect on N and E cadherins upon CMV infection needs to be tested.\n\nFigure 5G: IF images need to be quantified (similar to comments in 1F and 3F).\n\nFigure 6: The authors use an RNA-FISh strategy to examine HCMV TGFb and N-Cadherin expression in liver biopsies from BA patients. Although evidence that more CMV is present in BA samples is clear, it is not apparent that the CMV positive cells have more TGF-B and N-cadherin. Similar to previous comments, the IF signals need to be quantified.\n\nMore information also needed for qPCR method for detecting number of hCMV copies per cell.\n\nFigure 7: Figure 7G shows low expression of UL83 RNA, which encodes the major cytomegalovirus structural protein pp65, suggesting inefficient viral assembly if any. To support the claim of productive infection, the authors should provide gene expression data for different temporal kinetic classes especially for HCMV capsid proteins (e.g., UL86, UL85, UL46, UL48A) for cells in Cluster 5, currently absent in Figure S9.\n\nAccording to lines 358-359 (Figure 7H), approximately 9% of the total organoid cells are GFP-positive. However, this visually appears significantly overestimated when compared to the number of cells in Cluster 5 in Figure 7A. The authors should address this discrepancy and clarify their methodology for estimating GFP-positive cells.\n\nWhile Figures 7E-F provide interesting insights into HCMV's impact on cholangiocyte differentiation, the manuscript misses an opportunity for a detailed comparison between Cluster 5 and other clusters within HCMV-infected organoids. Such analysis could uncover factors driving Cluster 5's unique susceptibility to GFP and viral RNA expression, offering a more comprehensive understanding of host-virus interactions and infection heterogeneity.\n\nFigures 7B, 7C, and S8A show that cells in Cluster 5 express low levels of cholangiocyte-associated markers (e.g., KRT19, KRT18, SPP1, CLDN4, and CD24). An in-depth analysis of Cluster 5's gene expression profile is needed to determine the identity and differentiation state of these cells.\n\nThe methods section lacks key details about single-cell RNA sequencing. It should include information about the targeted cell recovery and the number of cells that passed quality filters, the number of genes detected across all cell types, specific QC thresholds applied during analysis etc.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Review of Ye et al\nThis manuscript by Ye et al entitled \"HCMV infection disrupts cholangiocyte organoid barrier functions by promoting TGF-B-mediated EMT\" seeks to expand our knowledge base concerning cytomegalovirus infection of neonates, with an eye on discerning whether HCMV could be an etiological agent involved in the development of biliary atresia (BA), a life-threatening disease due to cholangiopathy of the bile ducts. The authors utilize an exciting new organoid model of cholangiocytes to study the effects on growth and gene expression after HCMV infection. We know little about the ability of HCMV to infect these cells, and the authors are the first to really show that cholangiocytes can be infected, albeit at a very low level of permissiveness characterized by only a miniscule amount of viral shedding from what looks to be a very small proportion of cells within the organoid themselves. The authors delve into gene expression changes both using bulk RNA seq and single cell RNA seq on these infected organoids and (not surprisingly) find a dramatic number of changes after infection. They also examine several actual BA patient tissue samples to look for evidence of viral DNA within these tissues and whether or not some of the changes they see within the organoids with respect to gene expression hold true within these tissue samples. They try to make the case that HCMV infection is potentially driving the development and progression of BA via a TGFbeta driven transition to EMT. While some of the data is compelling and the utilization of a 3D platform for infection is exciting, this reviewer has several issues with the interpretation and presentation of data that need to be addressed. Major points where explanation/further analysis are warranted are outlined below.\n\nMajor points:\n1. On pages 3 and 4 the authors try to establish the connection between HCMV and BA development. Clearly, there is very little concrete evidence here and this is brought to bear by the literature cited saying there were HCMV positivity rates in BA studies that ranged from ~8-~77%. This is a ridiculously broad spread. Unfortunately, their examination of patient tissues was not tremendously more informative, landing at the higher end of this range (at 65.5%. However, the problem is that non-BA patients showed 35% positivity, thus leaving this association somewhat fuzzy.\n\n2. Figure 1:\nFig 1: This figure begins their analysis of infection in the cholangiocyte organoids. Fig 1A-C talk about the initial interpretations of these infected organoids with respect to size and shape and number. There are many things that need to be much more clearly defined in these experiments. First is the way in which the infections are performed. In the methods section on p 22, the authors describe an infection protocol where organoids are disassociated, presumably counted (although this is not explicitly stated), infected at an MOI=1, then asked to \"reform\" into organoids by resuspension in Matrigel. Several questions arise: are the mock organoids treated in the same way? I am presuming yes, but again, this is not explicitly mentioned. How long were the cells exposed to virus prior to rinsing and \"reforming\"? What percentage of cells are actually infected in this manner? You are infecting at an MOI=1, but do all cells take up the virus? This could (and should) very easily be discerned via fixing and staining the disassociated/infected cells at 1-2 hpi with an antibody to either pp71 or pp65 (tegument proteins). All cells should possess punctate spots of either of these proteins either within the cytoplasm or within the nucleus of all cells if the virus is capable of binding and entering these cells. This is incredibly important information, as it is clear from all the organoid images that are showing GFP positivity that not all cells are productively infected. Which leads to the question - what percentage of cells are GFP+ in these organoids? This must be explicitly stated somewhere.\n\nThis \"reforming\" of organoids and the subsequent lowering of numbers and size could all be due to cell death precipitated by the virus. This is not discussed or taken into consideration anywhere. Are cell numbers declining due to virus induced apoptosis or necrosis? This could easily be examined if instead of \"reforming\" organoids, the cells were allowed to stay in suspension as single cells (or as a monolayer)? Additionally, have these experiments been carried out for a longer period of time (the longest appears to be ~12 dpi). Is this time taken due to cell death/destruction of the organoids?\n\nThe problem continues when data is presented in bulk RT-qPCR and enumeration of HCMV DNA copies within the organoids. Again, how many of these cells actually harbor genomes, express transcripts and shed virions? Given the sparse localization of GFP signals (and the small proportion of cells in \"cluster 5\"), the answer is probably not very many.\n\nIn Fig 1E, the authors talk about taking the supernatant from the infected organoids and also seeding infected cells onto fibroblast monolayers. Information needs to be given regarding whether this supernatant was diluted and how many cells were seeded onto the wells of fibroblasts shown. As it is, this figure gives very little information except to see a minute quantity of virus is shed, presumably by a very few cells within the organoids.\n\nLastly, the immunofluorescence images in Fig 1F and G are not nearly high enough magnification to conclude that GFP+ cells are showing \"reduced expression\" of these markers. And are these whole organoid Z stack reconstructions? They do not appear to be single sections, but this needs to be explicitly stated. The choice of marker proteins to examine should also be explained.\n\n3. Fig 3: This figure focuses on the infected organoid's expression of EMT-associated genes (again, in bulk RNA format). The authors are exploring whether there may be a role of EMT in BA pathogenesis and whether infection could be promoting this phenotype.\n\nImportantly, the authors neglect the literature on HCMV and glioblastomas that has shown promotion of EMT in this pathogenesis of glia. Certainly, the RNA seq and confirmatory RT-qPCR data show quite clear changes occurring in the mRNA levels of these genes as called out (that are involved in EMT and in fibrosis).\n\nThe authors point out two proteins that are a signature for EMT - upregulation of N-cadherin (CDH2) and downregulation of E-cadherin (CDH1). The RNA data analysis certainly shows this trend. However, as presented in Fig 3F, the claims regarding GFP+ cells showing the most changes in the levels of these proteins cannot be substantiated. These images, once again, are much too low magnification to definitively say anything about co-localizing signals.\n\n4. Fig S3: This figure is troubling in several ways. First, why is EBV (which the authors claim is not infecting the cells) changing their size and shape similarly to HCMV? Secondly, CDH1 is not downregulated by HCMV infection in these experiments (as it was in Fig 3). Lastly, the IF images shown in S3H and I are just not convincing.\n\n5. Fig S4: This same phenomenon of mRNA increases for both CDH1 and 2 are seen with the BTS treatment, which the authors say causes EMT. However, once again the protein imaging that is presented in S4G does not show the predicted changes (neither E nor N-cadherin are increased in these images). Additionally, why is the Para treatment not behaving like predicted? No explanation is offered.\n\n6. Fig 4: Once again, the IF images showing ZO-1 staining are not at all convincing, much higher magnification is needed to discern specific apical versus basolateral staining. In addition, several papers have shown that HCMV infection causes loss of ZO1 integrity in different types of cells (these should be cited). Thus, this would not be a novel finding. The Rhodamine exclusion experiment shown in Fig 4C, while clear in the mock-infected organoids is not at all convincing in the infected organoids, once again due to the imaging conditions. Additionally, why is the HCMV-infected organoid pictured in the second row of panels so much smaller than that in the last row? The size bars are the same. Is there that much variation in size? These images would be more convincing with a single confocal slice through the middle of the organoid. In Fig 4 D and E, why are the mock and media control samples substantially different in their readings? This makes the interpretation of the TEER levels of the treated groups more challenging. Were these all done at the same time/conditions? Why do the CMV and EBV samples both behave the same, since the EBV samples were purportedly \"not infected\"/EBV DNA+?\n\n7. Fig 5: The authors convincingly show that in their infected organoids, TGFb is increased at both the mRNA and protein levels. Again, this is not surprising, given there are several papers that have shown this in different cell types after HCMV infection (these should be cited). Once again, however, these data are inconsistent with those presented in Fig 3 with respect to CDH1/E-cadherin changes. Here, there are essentially no changes at either the mRNA or protein level after infection. Additionally, it would be very informative to see what percentage of cells were GFP+ in the HCMV versus HCMV+SB organoids - they appear to be dramatically different.\n\n8. Fig 6: These images once again are not high enough magnification to make definitive calls regarding colocalization of signals. Frankly, the images shown in Fig S6 are dramatically more convincing and should be included in the main figures.\n\n9. Fig 7: This single cell RNA seq data clearly shows differences between the infected and uninfected organoids and there are clear \"shifts\" between the percentages of cells within the 5 different clusters. It is also clear that all clusters have some viral gene expression, although only cells within cluster 5 appear to be fully infected and expressing GFP to any level. This brings me to the crux of the problem: what percentage of the cells within each cluster are actually expressing virus and viral proteins/mRNA!! You should be able to tell this from your single cell data. Yes, cells within all the clusters express viral mRNA - but what actual percentage of those cells are expressing? And do all the other cells have latently infected genomes?? Or are they real bystander cells??\n\nImportantly, certainly there are shifts in the proportions of cells within the clusters, but this doesn't mean actual viral protein/RNA expression is what's driving this shift. Additionally, and very importantly, what constitutes GFP+ vs GFP- cells? How are these gated?? A recent paper studying RNA seq changes in cerebral organoids from the Terhune lab showed that there was not really a \"GFP-\" population, but instead, a GFPlow population of cells. This must be clearly defined here.", "reviewer_4": "General comments:\n• The authors described in vitro experiments examining whether there is an association between CMV and neonatal biliary atresia (BA) They cultured human iPSC to produce a cholangiocyte-like cell organoid model infectable and infected with HCMV strain TB40E containing and eGFP reporter. Infection resulted in changes in organoid growth, structure, and bulk RNA-sequencing showed increased EMT pathway expression. Using TGF small molecular inhibitor (SB431542), which is also an Activin inhibitor, they interpret this TGS-B effect as central to the observed phenotyp. Using bulk and single cell RNA-seq, they suggest cluster of HCMV infection, and that such infection directly causes EMT, and hence biliary atresia. They then examined tissue from an uncertain number of patients with biliary atresia.\n• The title is misleading – the evidence for TGF mediated change on EMT being the major, only or even important effect is not provided by the results. This should be changed.\n• It appears this model is permissive to HCMV infection but does not allow for the full replication cycle given the decrease in HCMV DNA copies from 5dpi to 12 dpi. It would be interesting to provide immunofluorescence data to show IE/E, pp65, and pp28 staining to confirm this.\n• The discussion is very long and could be made shorter by removing some repetition of the results.\n• Keep the labelling and description in your figure legends consistent. The letter in your figure legends should be written before each description. This is currently not consistent throughout each figure.\nSpecific comments:\n• The way the paper is written does not help understand the authors work. There are too many additional words at the start of many sentences that make this manuscript difficult to read (e.g. moreover, in addition etc.). The abbreviations are not used consistently e.g. PI or P.I. The terminology around the model is inconsistent throughout the manuscript. At times is it referred to as “cholangiocyte-like cell organoids” and others as “cholangiocyte organoid model”.\nIntroduction\n• L55 It is important to somewhere explain why most neonates do not have biliary atresia (it is rare), yet breast milk contains HCMV in more than 50% of births.\n• L68 Asserting that HCMV contributes to biliary atresia is untrue – there are data suggesting 5-10% of patients may have HCMV contributing, but examining those data, and the reference provided, demonstrates this is speculative and in need of much further study.\n• The first paragraph of the introduction is too long, and therefore the importance of this study is lost. Removing extra information that is not relevant to this manuscript would improve this. For example\no Lines 60-65 should be summarised\no Line 66 is repeated\n• Integrate references in line 75.\n• L84 It is misleading to write HCMV has two modes of infection. HCMV may cause productive/permissive infection either on primary or reactivation from latency.\n• Lines 78-81: Make it clearer that these studies are showing an association between HCMV infection and BA developments in infants.\n• Lines 84-89 should be a separate paragraph.\n• Line 100 would benefit from explaining why this model was used and why it is described as “cholangiocyte-like cell organoids”\n• Line 102 is unclear, please clarify.\n• L109 In the absence of a control population, these human data are difficult to interpret.\nResults\n• Line 121: A percentage of the number of infected cells would be helpful rather than an explanation that not all cells were infected.\n• Line 122: Please clarify what you mean by “the number” of organoids were similar?\n• L129 Which LATs were detected?\n• L128 Genes of HCMV, as dsDNA virus are not detected through RT-PCR.\n• L131 How was HCMV quantitated in organoids?\n• Line 163: Specific upregulated/downregulated pathways of interest should be induced e.g. pathways associated with liver disease/function.\n• Line 172: Is this enrichment score significant?\n• Line 281-283: How does this compare with other publications?\nDiscussion\n• Line 370: Should include what the significance of this study is.\n• Line 425: Should say “sonic hedgehog”\n• L428 HCMV has also been shown to affect SHH signalling (Egilmezer et al 2024). The term “mediate” here is uncertain and arguably incorrect.\nMethods\n• The TGF-B inhibitor used also inhibits Activin via effects on ALK4, ALK5, ALK7. Does this affect the interpretation of results in relation to TGF-B?\n• L503 The O’Connor 2012 reference is not correct for TB40E. The viruses exist as different types – the authors should define which TB40E they used and it’s detailed characteristics in the methods.\n• Line 528: There is no indication of when the authors are describing the method for organoid generation. This should be a separate paragraph, with the previously published methodology clearly referenced and a brief summary of the organoid generation protocol.\n• Line 549: Was this using a previously published protocol? Please provide the reference or include more descriptions.\n• Line 552: Do not use abbreviations without defining their meaning first e.g. “RT”\n• L603 Is 8 days culture sufficient for mature biliary organoids?\nFigures\n• Figure 1: Include the letter label in the figure legend before the description. It is written after in F and G\n• Please keep abbreviations consistent e.g. P.I. in figure 4\n• Figure 2B: This heat map may be better off included as a supplementary figure as it does not provide specific data that is relevant to the paper.\n• Figure 4: Include the letter label in the figure legend before the description. It is written after in D and E\n• Figure 5: Fix letter labelling as explained for figure 1 and 4. The level of TGF-B is expressed relative to mock. It would also be good to compare the expression in these cells vs other models, given inhibition of TGF-B will be of baseline, as well as of HCMV induced increase.\n• Figure 7: Figure legend is confusing and does not correlate with the figures from D onward.\n• Figure S3: It is unsurprising EBV was not cultured in organoids, and these data are not germane to the paper.\n• Figure S4: The release of free TGF-B is of interest, although tissue fixed TGF is of more interest.", "reviewer_5": "The authors describe their study on biliary atresia (BA) using iPSC-derived cholangiocyte organoids and how infection of human cytomegalovirus (HCMV) affects the cholangiocyte function. The authors show that the TGF-b pathway is involved in this, which might have implications for future therapeutic intervention.\nGeneral: This study shows a great example of how cholangiocte organoids can be used to study relevant disease, in this case BA. By infecting them with the actual HCMV virus, this model allows the study of pathways involved. Although it being a relevant and important study, I do have several issues with it. In general, the text needs to be revises substantially.\nGenerally, I wonder if cholangiocyte organoids are the right model (you need cholestasis and fibrosis which is not modeled in this single epithelial cell culture. Maybe the authors can be an advocate for this model and explain a bit more why this model is the best to study BA.\nDid you analyse the cholangiocyte function after differentiation? If so, how? And how does this relate to native cholangiocyte function? And, did all iPSC lines behave the same?\nOther focus points:\nThe summary needs more explaining: \"... through TGF-b/EMT, which has ...\" is not clear what is meant by TGF-b/EMT.\nIntroduction:\n- I think BA needs to be better introduced. There are different types of BA, and to me it is not clear what type the authors are modeling. Also it should be clear that not all BA is related to HCMV infection.\n- there are several textual mistakes. While here and there the text could be more clear and concised, the authors mention \"fibrosis of epithelial cells\". Cells do not get fibrosis, it is the extracellular matrix (ECM) that gets stiffer and ECM proteins more abundantly present.\n- in line 69, it states \"...of certain BA patients (6).\" What do you mean by 'certain' - be more clear in what you mean.\n- in line 70, you mention congenital disease, I guess (but I am not sure) this needs to go through infection via the mother then? How are you modeling this in the organoids?\n- line 76 mentions '..that infants with...'; just a question, did all infants have elevated antibody levels? In other words is in all cases of BA, HCMV present? make this a bit more clear.\n- line 80, what is meant by \"positivity rates ranging from 7.6% to 76.9%\"? I do not know what the percentages represent and how important this is to know. The end of the sentence is also quite vague, mentioning \"using various methods and tissue samples (13).\" this is not useful information like this. What methods, what tissues? Be more specific when describing these facts.\n- in the introduction, the authors mention that they performed single cell RNA sequencing and found 5 clusters of cholangiocytes, one of which fully supported HCMV lytic infection. I missed where this knowledge came from. There is no reference to a publication, nor is it explained further in the manuscript. Please enlighten me.\n- I miss a clear aim / hypothesis of the study\n- I think the conclusion, as mentioned in line 112, that the results have proven to show 'direct contribution' of HCMV infection to BA, largely overstated. I do not think the data that is shown is providing this.\nResults:\n- Figure 1S is abundant. This has been published before and can be removed.\n- Fig 1A: it is not clear if there is GFP signal in the cells. It would be good to provide larger images and include co-localization with nucleus and/or cell membrane. It seems that the GFP signal is in the lumen of the organoid, which means that is then is not inside the cells. I also would like to see the infection efficiency (so were all organoids infected, or some? And were all cells in an organoid infected, or just a few? I would also like to see an overview of the total organoid culture. We only see 1 organoid now, which might not be representative to the rest of the culture. Furthermore, I am not sure if you can say that the morphology of the organoids after infection is changing. Whit differentiation, all organoids create a thicker wall and exhibit irregular shapes. And finally, did you include mock transductions, with only GFP? How did these organoids look and behave?\n- in line 132 you mention to measure 30 copies of DNA per cell, but in the methods section, you mention that you aim for an MOI 1. This then does not make any sense, as you expect on average 1 copy per cells then, right?\n- the Hs27 line is not introduced in the methods section. Why this line, and how was it grown/used in this study?\n- In figure 2 the bulk RNA sequnencing data is discussed. Can the authors explain why they find hepatocyte development-related genes? Isn't this not providing evidence that the organoids are not fully differentiated cholangiocytes? Did the authors even assess cholangiocyte functionality and related this to primary cholangiocytes?\n- I would love to hear more in depth analysis about the EMT - is this real EMT?E and N-cadherin expression alone do not really prove this. I think this is a overstated conclusion and should be investigated further.\n- Overall, the result section needs a bit of textual revision to make it more clear.\n- I suggest the authors to critically look at the figures and clarity of the figures. Larger images, overview of a larger part of the cultures instead of showing just the one organoid, would help."}, "author_response": "Authors: Thank you for the valuable comment. We agree that was an oversight to not quan7fy the data. In this version of the manuscript, we have quan7fied the immunofluorescence signal intensi7es and Western blot band intensi7es in the data we show.\n\nThe manuscript does not adequately describe how cholangiocyte organoids were infected. For example, it is unclear if spin infec7on was used or if the mock infec7on protocol was iden7cal. How long was the infec7on? Was it in suspension, in a pellet or in a plate? These details should be included for reproducibility.\nAuthors: Thank you for the comment. The details are now added to “HCMV infec7on of cholangiocyte organoids”. Briefly, the infec7on occurred for 3 h, cells were dissociated from the organoids, inoculated with HCMV, and placed sideways in a cell culture incubator with gentle tapping every 1 h.\n\nIt is implied that the model supports HCMV replica7on and viral produc7on - referring to the single cell RNA sequencing data, the manuscript frequently men7ons that the model demonstrates \"cells with high levels of viral replica7on\" (line 347) or \"producing new viruses for transmission.\" However, this claim overstates the evidence presented. GFP expression alone is not sufficient to confirm the produc7on of infec7ous virions necessary for transmission. To defini7vely demonstrate that cholangiocytes support produc7ve HCMV infec7on, the authors would need to use the supernatant from infected organoids, which should contain infec7ous virions if replica7on is complete. This supernatant could then be used to infect other organoid suscep7ble cell line models to confirm produc7ve infec7on.\nAuthors: Thank you for the comment. We performed the experiment using supernatants from HCMV-infected CLC organoids added to produc7ve infec7on permissive human fibroblasts, where this resulted in GFP+ cells (Figure 1j). Moreover, we FACSorted GFP+ cells from infected CLC organoids and applied them to human fibroblast, which also resulted in GFP signals (Suppl. Figure S1b). These experiments confirmed that produc7ve infec7on of HCMV occurs in infected CLC organoids, par7cularly the GFP+ cells.\n\nWhat is the percentage of infected organoids or percentage of infected cholangiocytes per all cells that are obtained with this model? From Fig 1F and 1G, the infec7on efficacy does not seem to be very high. The authors should dissociate the organoids to single cell level and quan7fy infec7on rates together with other relevant markers by Flow cytometry.\nAuthors: Thank you for this comment. To understand the infec7on rate of HCMV in the cells of CLC organoids, we performed several experiments. First, we examined the infec7on rate based on GFP signals by flow cytometry, which was below 10% (Figure 1 d,e). However, qPCR data showed GFP- cells also contained HCMV DNA (Figure 1f), which we probed using ISH; the rate was found to be around 20-30% (Figure 1g). An an7body against pp28 was also detected in approximately 10-15% of cells (Figure 1h). Together, there were around 20-30% of cells infected by HCMV in the CLC organoids, where some support ly7c infec7on and some do not.\n\nLine 124 – 125 Figure 1 A states that the morphologies were different and organoids had a thicker boundary. This is not very clear in the representa7ve image. Can the average length of the boundary be measured and quan7fied with numbers?\nAuthors: Thank you for this comment and sugges7on. We took mul7ple images and examined the thickness of the organoid boundary in the mock, HCMV-GFP- and HCMV-GFP+ organoids. On average, the mock organoids had a mean boundary thickness of 0.50 µm with a standard devia7on (SD) of 0.13 µm. The GFP+ organoids were found to be 1.50 ± 0.67 µm, and GFP- organoids had 1.22 ± 0.94 µm. These data are added to the text in the first results paragraph.\n\nFigure 1D: Why are the copies per cell decreasing over 7me? In addi7on, the control shows copies in fibroblast cell aper 48 hours. This should also be quan7fied at a later 7mepoint to see if the viral counts go down because of lysis of infected cells. This could also serve as a reason for decrease in copies in the organoid model. If not, then viability of organoid needs to be measured with 7me. This graph also needs an appropriate nega7ve control such as a heat-inac7vated virus.\nAuthors: Regarding this comment, the decrease in the DNA copy number over 7me in the previous presented data was due to the general lost of organoids over 7me in the culture. We measured the viability of the organoids using Annexin-V and propidium iodide and found that the GFP+ cells had a higher rate of cell death at ~10% compared with the GFP- cells or mock cells at <5% (Suppl. Figure S2 a,b). We have presented the data without the 7me points because it derails from our message to show that HCMV infec7on can occur as ly7c and non-ly7c modes, which we now compare in Figure 1f.\n\nFigure 1F and G is not very convincing by the image provided. It is not apparent that the signal for CK19 and SOX9 are lower in infected organoids. This needs to be quan7fied by measuring the intensity of the IF signal.\nAuthors: Thanks for poin7ng this out. We agree that the immunofluorescence images are not very clear here and the point we are trying to make is that HCMV infec7on can bring about changes to the differen7a7on state of the cells as cholangiocytes or hepatocytes. However, the main message of the manuscript is to demonstrate that HCMV infec7on can induce EMT, instead of triggering the changes in the cell state. Thus, we have moved the data to before Figure 6 as Suppl. Figure S4a,b in the revised manuscript. The data was also analysed for fluorescence intensity for CK19 and SOX9. HCMV-GFP- and HCMV-GFP+ cells both had decreased CK19 expression, with a more significant reduc7on in the lauer. SOX9 was significantly decreased in both the HCMV-GFP- and HCMV-GFP+ cells.\n\nFigure 2 and 3 represent data from the bulk RNA sequencing analysis comparing infected and uninfected cholangiocyte organoids and should be merged into one figure. Figure 2D and 3C should be presented in supplementary. Figure 2B and 3B can be merged. Figure 2E, 3D and 3E are valida7on of DEGs from the RNA-seq experiment. They can all be merged into one supplementary figure.\nAuthors: Thank you for the sugges7ons. We have merged the different panels of Figure 2 and 3 together while puvng the rest in Suppl. Figure S3. Now Figure 2 shows the analysed bulk RNA-seq data to demonstrate the difference between HCMV infec7on for cholangiocyte/hepatocyte markers, followed by EMT-related genes, such as the upregula7on of CDH2 (N-cadherin). Changes in E- and N-cadherin at the protein level were validated using Western blovng and immunofluorescence. Overall, this figure should deliver the point more clearly according to the sugges7ons by the reviewer.\n\nFigure 2D and 3C: Is this data from the RNA seq? Or validated in the same organoid models and donors? If so, the number (N) needs to be indicated in figure legends and individual points need to be indicated on the graph.\nAuthors: Thank you for poin7ng this out. These data are from the bulk RNA-seq data from n=5 biological replicates. The informa7on is added to the figure legends.\n\nFigure 3F: This is the only func7onal readout of EMT transi7on presented, and similar to previous comment on Figure 1F conclusions cannot only be made based on one representa7ve microscopic image. The intensity of the signal needs to be quan7fied and other func7onal readouts of EMT transi7on at protein level needs to be measured. Using flow cytometry strategy as discussed above, the authors should examine the expression levels of E and N cadherin by staining with an7bodies and measuring differen7al expression at the protein level in the GFP posi7ve (infected) and GFP nega7ve (uninfected) cholangiocytes within their organoids.\nAuthors: Thank you for the comment. Instead of flow cytometry, we decided to perform addi7onal immunflourescence and 7le scans to beuer visualize the GFP+ and GFP- cells in the HCMV infected organoids. As men7oned above, we quan7fied the band intensi7es from Western blots of E- and N-cadherin in the mock and HCMV-infected CLC organoids (Figure 2d) and fluorescence intensi7es from the organoids (Figure 2f,g and Figure 4h,j) and 7le scans (Suppl. Figure S9).\n\nFigure 4, Viability of organoids needs to be measured aper infec7on. From the representa7ve images it appears that organoid viability is being severely compromised and this could explain the loss of barrier func7on.\nAuthors: The viability of infected organoids was measured using Annexin-V and PI using flow cytometry (Suppl. Figure S2 a,b). As described above, HCMV-infected CLC organoids showed ~10% of cells undergoing apoptosis, which is unlikely to be the major factor in determining the loss of barrier func7on.\n\nThe authors should also examine cholangiocyte func7onality such as via alkaline phosphatase ac7vity in presence and absence of HCMV infec7on both at early and later 7mepoints, to gain more insight into the effects of HCMV infec7on on cholangiocytes.\nAuthors: Thank you for the sugges7on. We performed the measurement of ALP ac7vity on day 8 and day 12 PI and found significant decreases in the HCMV infec7on compared with the mock sample (Suppl. Figure S5b). This data adds to the evidence that cholangiocyte func7onality is decreased upon HCMV infec7on.\n\nFigure 5: In general the authors need to quan7fy IF images and western blots (how many 7mes was this performed?), par7cularly those measuring N and E cadherin since this is one of the main markers for EMT transi7on inves7gated.\nAuthors: Thank you for the comment. We have quan7fied the immunofluorescence images and Western blovng data and presented them as column graphs throughout the manuscript. In par7cular, 7le scans were performed to allow for the measurement of the E- and N-cadherin intensi7es for all the organoids in the culture. We believe this will allow for a beuer understanding of the experiment.\n\nFigure 5A: Did the authors also see TGF-B expression upregulated in their RNA-seq dataset (that was at 10 dPI). If not, do the authors have an explanation about why this was not observed in their RNA-seq.\nAuthors: Thank you for this comment. We have re-visited our scRNA-seq data and found that the TGFB1 gene expression was upregulated in the HCMV-GFP- cells and the HCMV-GFP+ cells when compared with the mock sample (Figure 5k). RT-qPCR also validated this data, as shown in Figure S4a. The increase in TGF-b protein also occurred in the ELISA assay (Figure 4b), as well as the Western blovng (Suppl. Figure S6 b-d).\n\nFigure 5B: The western blot needs to be quan7fied from at least 3 separate blots from 3 independent experiments with different donors to make a statement that N and E cadherin levels are being up or down regulated.\nAuthors: Thank you for the sugges7on. We have done so for all the Western blovng data from at least three independent experiments. The band intensi7es compared with the b-ac7n are shown in various figures.\n\nLine 252: Why is SB causing decrease in expression of CMV genes? Is the reversal of EMT phenotype actually controlled by TGF-B signalling or by the CMV genes? Is the inhibi7on of TGF-B signalling also causing inhibi7on of CMV genes? If so, are the CMV genes resul7ng in EMT transi7on, not necessarily via a TGF-B reversal pathway?\nHow do CMV induced genes dysregulate TGFB signalling and why inhibi7ng TGF-B inhibit the CMV gene expression, which is the chicken and which the egg. The authors should speculate/discuss.\nAuthors: Thank you for the ques7ons. It is a puzzling ques7on as to why SB431542 would appear to affect HCMV gene expression. To address this ques7on, we used a blocking an7body against TGF-b and repeated the experiments. We found that an7-TGF-b an7body also reversed the induc7on of EMT genes expression (Suppl. Figure S6), but the level of viral gene expression was not affected. Thus, this is probably associated with the SB431542 inhibitor affec7ng viral gene expression, although the mechanism is not clear. One recent study showed that SB431542 could inhibit SARS-CoV-2 N gene expression in Vero cells but the authors did not put emphasis on this (Verma et al., J Virol 2025). Since an7-TGF-b did not affect HCMV gene expression, it likely means that there is an intrinsic pathway that is associated with viral gene expression or replica7on. As suggested by reviewer 4, SB431542 could also inhibit the ac7vin pathway, which is closely associated with the TGF-b signalling pathway. Along this line, another study showed that inhibi7on of the ac7vin pathway could reduce SARS-CoV-2 replica7on, though it is not clear whether viral genes are suppressed (Merzger et al., Viruses 2022). If inhibi7on of TGF-b or ac7vin can limit HCMV gene expression or replica7on, then it indicates a mechanism that has not been shown for HCMV in cholangiocytes, which may be worth inves7ga7ng in the future.\nTo the reviewer’s ques7on, if the an7-TGF-b blocking an7body does not hinder HCMV gene expression but abrogates HCMV-induced EMT, then it would suggest that it is the TGF-b induced by HCMV that is media7ng the process. However, we cannot rule out completely whether the HCMV genes are capable of inducing TGF-b and/or EMT directly. This could happen in a different subtype of cells, as eluded in the scRNA-seq data where Cluster 3 in the infected CLC organoids could mediate the upregula7on of CDH2 in Clusters 1 and 2. These points are added to the discussion.\n\nFigure 5: SB effect on N and E cadherins upon CMV infection needs to be tested.\nAuthors: Due to the above that SB431542 could influence HCMV genes, and the fact that an7-TGF-b blocking an7body could achieve similar effects, we performed the analysis of N- and E-cadherin expressions using immunofluorescence and confocal microscopy. We performed 7le scans of the CLC organoids and found that the blocking an7body can abrogate the HCMV-induced N-cadherin expression. E-cadherin remains slightly decreased when comparing HCMV-GFP- cells and mock cells (Figure 4 h-j), as shown in the 7le scan in Suppl. Figure S9.\n\nFigure 5G: IF images need to be quan7fied (similar to comments in 1F and 3F).\nAuthors: All immunofluorescence images were quan7fied for the proteins of interest, i.e., E-cadherin, N-cadherin, a-SMA, S100A4, and ZO-1.\n\nFigure 6: The authors use an RNA-FISh strategy to examine HCMV TGFb and N-Cadherin expression in liver biopsies from BA pa7ents. Although evidence that more CMV is present in BA samples is clear, it is not apparent that the CMV posi7ve cells have more TGF-B and N-cadherin. Similar to previous comments, the IF signals need to be quan7fied.\nAuthors: Thank you for the comments. We have since quan7fied the immunofluorescence signals in BA liver 7ssues and non-BA liver 7ssues from the 7le scans. Cells with or without HCMV-DNA were measured for the fluorescence intensi7es for N-cadherin and TGF-b in both the BA and non-BA liver samples. However, HCMV-DNA could not be detected in two of the non-BA samples and the expression of the molecules remained rela7vely low. The data are now shown in Figure 6c,d.\n\nMore informa7on also needed for qPCR method for detec7ng number of hCMV copies per cell.\nAuthors: Thank you for men7oning this. Addi7onal details about this qPCR method are now added to the Methods Sec7on.\n\nFigure 7: Figure 7G shows low expression of UL83 RNA, which encodes the major cytomegalovirus structural protein pp65, sugges7ng inefficient viral assembly if any. To support the claim of produc7ve infec7on, the authors should provide gene expression data for different temporal kine7c classes especially for HCMV capsid proteins (e.g., UL86, UL85, UL46, UL48A) for cells in Cluster 5, currently absent in Figure S9.\nAuthors: Thank you for the sugges7on. We have revisited the scRNA-seq data to examine HCMV gene expression among the 5 clusters based on the temporal kine7c classes, which can be found in Suppl. Figure S10. UL85 and UL86 can be found in TC6, UL48A in TC3, and UL46 in TC7, but these genes did not show any expression in Clusters 1 to 4. Their expression, however, was high in Cluster 5.\n\nAccording to lines 358-359 (Figure 7H), approximately 9% of the total organoid cells are GFP-posi7ve. However, this visually appears significantly overes7mated when compared to the number of cells in Cluster 5 in Figure 7A. The authors should address this discrepancy and clarify their methodology for es7ma7ng GFP-posi7ve cells.\nAuthors: Thank you for the poin7ng this out. To closely understand the level of infected GFP+ cells, we performed addi7onal experiments. From flow cytometry, the percentage of GFP+ cells was approximately 10% (Figure 1 d,e), which correlated to around 12% measured by immunofluorescence of pp28 (Figure 1 h,i). However, the percentage of pp28+/GFP+ cells was about 5% in the infected organoids, which indicates viral replica7on. The scRNA-seq data was generated from mock and FACSorted HCMV-GFP- and HCMV-GFP+ cells, which aper data processing and filtering, the numbers may not necessary represent the crude percentage of GFP+ cells. Also, in the previous version of the analysis, the ~9% GFP+ cells were from the HCMV-GFP- and HCMV-GFP+ datasets mixed together and it is agreeable that data processing and filtering may affect the end result as well. Thus, the addi7onal experimental data in Figure 1 in the revised manuscript makes it more clear on the percentage of GFP+ cells following infec7on.\n\nWhile Figures 7E-F provide interes7ng insights into HCMV's impact on cholangiocyte differen7a7on, the manuscript misses an opportunity for a detailed comparison between Cluster 5 and other clusters within HCMV-infected organoids. Such analysis could uncover factors driving Cluster 5's unique suscep7bility to GFP and viral RNA expression, offering a more comprehensive understanding of host-virus interac7ons and infec7on heterogeneity.\nAuthors: Thank you for the comments. Our previous analysis, which is also displayed in Figure 5b and Suppl. Figure S11, show that Cluster 5 expresses more of the cholangiocyte markers, and with top 5 DEGs as HES6, DDIT3, GADD45G, MAP1B, and PCSK1N. However, these genes belong to different pathways and they likely point to responses for cells undergoing ly7c infec7on, such as stress, DNA damage, and change in microtubules. This is indeed different to Clusters 1 to 4. Clusters 1 and 2 appear to express genes involved in 7ssue development, and cilium assembly and organisa7on, respec7vely (Suppl. Figure S13). Clusters 3 and 4 have genes that are associated with hormones and metabolic processes, respec7vely (Suppl. Figure S13). Meanwhile, Cluster 5 mainly showed viral genes at a high magnitude compared with the host genes, which is indica7ve of viral replica7on.\nWe also analysed the the expression of possible entry receptors among the clusters, including NRP, CD44, EGFR, CD14, PARP1, FOSL1, and NRP2, but none of these stood out for Cluster 5 when compared with the other clusters that are uniformly distributed (data not shown). Therefore, the entry receptors may not dictate the heterogeneity of the clusters and possibly something to do with the cellular differen7a7on or environment that can be abused by HCMV infec7on, leading to the different onset of Cluster 5 from the other clusters. Pseudo7me analysis may indicate this, where in the HCMV infected CLC organoids, a trajectory can be seen from Cluster 1 or Cluster 2 leading to Cluster 5 (Suppl. Figure S12).\n\nFigures 7B, 7C, and S8A show that cells in Cluster 5 express low levels of cholangiocyte-associated markers (e.g., KRT19, KRT18, SPP1, CLDN4, and CD24). An in-depth analysis of Cluster 5's gene expression profile is needed to determine the iden7ty and differen7a7on state of these cells.\nAuthors: Following from the above response, the expression of cholangiocyte-associated markers KRT17, KRT19, and SLC4A2 were lower than the other clusters, while KRT18, CLDN4, and SPP1 appear to be similar if not higher than the other clusters, as shown in the revised Figure 5b,c and Suppl. Figure S11. Looking at Figure 5b, the expression pauern of gene markers resembles Cluster 1, which relates to the pseudo7me analysis, where Cluster 5 may have followed differen7a7on from Cluster 1. To note, the HCMV genes had significantly higher expressions than the top 5 DEGs (HES6, DDIT3, GADD45G, MAP1B, and PCSK1N), which suggests that Cluster 5 underwent ly7c infec7on. From these comments, it appears that ly7c infec7on in Cluster 5 was not due to a subtype of cholangiocyte that expresses entry receptors that prefer HCMV infec7on, and hence, result in ly7c infec7on, nor is it due to a unique cellular signature to support the replica7on cycle of the virus. Rather, it could arise from the infec7on of other clusters and become differen7ated over 7me. An indicator is that aper the HCMV infec7on of CLC organoids, GFP+ cells can be seen from 24 h PI, which would increase over 7me. A 7me course experiment with scRNA-seq from a few hours aper infec7on to day 11 PI would provide more in-depth findings to determine how Cluster 5 came about.\n\nThe methods sec7on lacks key details about single-cell RNA sequencing. It should include informa7on about the targeted cell recovery and the number of cells that passed quality filters, the number of genes detected across all cell types, specific QC thresholds applied during analysis etc.\nAuthors: This informa7on is now added to the Methods Sec7on."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I would like to commend the authors for a thorough revision of their manuscript. They have addressed my concerns by the suggested clarifications, quantifications and re-analysis of data, as well as by performing the additional suggested experiments that has strengthened the conclusions of the manuscript.", "reviewer_3": "Overall, the premise and major results of this resubmission are greatly improved and the authors have done a large amount of additional experimentation to satisfy the reviewers' comments and should be commended for this effort.\nI think the major conclusion as stated at the end of the introduction, that CMV can directly infect cholangiocyte organoids and induce TFGb/EMT pathways is justified by their data. Unfortunately, I think a lot of their extra efforts in trying to define the infection parameters with respect to gfp+/gfp- cell populations is creating more confusion/problems than it may be worth…. \nThe main issue is this: they show in figure 1g a FISH where they have used a viral-specific probe to assess their infected organoids. They show ~30% of the cells are FISH/genome+ - but of this population, only ~10% are also gfp+ (and actively shedding virus and making viral proteins). So that means another 20% of the cells have genomes (at low copy numbers) and according to their RNA seq data are expressing a limited set of viral genes. However, the MAJOR problem is that within these organoids, 70% of the cells have no sign of viral proteins/genomes! In and of itself, this reviewer doesn't have a real problem with this…. some cell types are not hugely permissive for infection…. \nHowever, the real problem is that when they are grouping their infected cells into \"gfp+\" (which is a very small proportion of the population) and gfp- (which is essentially 90% of the organoid cells), they are in essence \"handicapping\" the gfp- population, because the LARGE majority of those cells are uninfected and presumably behaving a lot like their mock infected cells. This certainly looks to be the case when you look at the single cell analysis – the distributions of cells and expression of marker proteins examined looks much more like mock infected organoids because they are 70% mock-infected cells!\nDespite this \"disadvantage\", there still are clear differences in some of the parameters that are examined, however, this reviewer thinks it is very difficult to interpret data presented as \"gfp-\", when it is in essence a heavily weighted uninfected population.\nPerhaps the more appropriate way to show the data is to use the gfp+ data that has been generated (which has clear distinctions from uninfected organoids) and the \"infected organoids\" (which is clearly a mixed population of cells, but shows very clear distinctions) versus the uninfected/mock organoid populations.\nI think the conclusions they are trying to make are more clear after much more analysis…. this reviewer just thinks the presentation would be more convincing without this gfp- distinction.\nShow that there is a mix of cells within the infected cultures, then leave it at that. The changes observed are clear….infection is making a difference.\nPresented data that is still is a bit problematic:\n1. The gfp- data in supplemental fig 4 is not convincing.\n2. The CFTR data in fig 3a and b is not that convincing, as there appear to be many more infected organoids measured and the spread of the mock organoids is very large.\n3. The interpretation of the KRT19 expression in fig 5b on p13 is not what is seen in the figure.\n4. The data presented in Fig 6b does not clearly show HCMV DNA in the nucleus….the magnification is just not high enough.", "reviewer_5": "The authors extensively adapted their manuscript with the comments and suggestions all reviewers made. When focusing on the replies to my own comments, questions and suggestions, I appreciate the clear responses and adaptations made to the manuscript. I do not have any further remarks."}, "author_response": "Authors: We thank this reviewer for the comments to enhance our manuscript. We are delighted to have addressed your concerns.\n\nAuthors: We thank the reviewer for raising this important point. We agree that infection does not occur in all cells within the HCMV-infected organoid cultures; our data indicate that approximately 70% of cells remain uninfected. Despite this, we observed significant differences in these cells compared to mock-infected or GFP+ cells across various experiments.\n\nIn light of the reviewer's comments, we have carefully reconsidered our data presentation. While the GFP- population contains both latently infected and uninfected bystander cells, we note that this population as a whole exhibits changes potentially attributable to the infection milieu. However, we acknowledge that this interpretation has a limitation: the observed alterations in bystander cells could result from signals originating from either GFP- (latently infected) or GFP+ (productively infected) cells. Currently, it is not feasible to definitively distinguish latently infected from uninfected bystander cells within the GFP- population for more granular analysis. As part of our future work, we plan to identify specific viral genes as markers for latency. Using targeted probes or antibodies, we will enable more in-depth analysis of latently versus productively infected cells. This will allow us to define the specific cell types that support each infection state and to further understand the distinct effects on bystander, uninfected cells within the infected niche.\n\nTo address this, we have modified the manuscript to use the following, more precise terminology:\n• Uninfected control = Mock\n• HCMV-infected organoid cells (non-GFP+) = Inf.\n• Infected GFP+ cells = Inf. (GFP+)\n\nThese definitions, now clarified in the first paragraph of the Results section, more accurately represent the populations we can currently resolve. Future work will focus on identifying specific viral gene markers (e.g., via probes or antibodies) to enable precise isolation and analysis of latently infected cells, productively infected cells, and true bystander uninfected cells. This will allow us to dissect their distinct roles and interactions, including defining which cell types support latent versus productive infection.\n\nIn response to the reviewer’s point regarding the GFP- population in our HCMV-infected organoid culture, and perhaps to satisfy the curiosity of the reviewer and ourselves, we performed a deeper characterization of these infected (Inf.) cells. To better distinguish between states of infection, we segregated GFP- cells into two subpopulations: those expressing any detectable viral gene (VG) and those with no viral gene expression (noVG). Out of 5,129 GFP- cells analyzed, only 213 (~4%) displayed any viral gene expression (VG cells). We have revised Suppl. Table S1 to focus specifically on this VG subset, as we agree that presenting data from all 5,129 cells would overestimate the proportion of latently infected cells in the culture. Based on our imaging data (Figure 1), approximately 20% of cells harbor HCMV-DNA, which would correspond to ~1,026 latently infected cells. The 213 VG cells therefore represent about 20% of the latent pool expressing at least one viral gene, albeit at markedly lower levels than productively infected cells (Suppl. Figure S14). The remaining noVG cells likely consist of either latently infected cells in which viral gene expression is minimized - consistent with established features of HCMV latency - or truly uninfected cells.\n\nWithin the VG subpopulation, we identified the viral genes most frequently expressed. UL22A and UL30 were detected in 14.08% and 13.15% of VG cells, respectively, representing the most prevalent viral transcripts (Supplementary Table S1). These genes are promising candidates for future use as markers to identify HCMV-infected cells in patient liver biopsies. Known functions of UL22A and UL30 are summarized in Supplementary Table S3.\n\nMoreover, we sought to identify host differentially expressed genes (DEGs) that could distinguish latently infected cells from uninfected cells within the HCMV-infected organoids. Comparative analysis of the VG and noVG subpopulations revealed that UBP1 (Upstream Binding Protein 1) was expressed in 55.4% of VG cells, at levels 1.5-fold and 1.3-fold higher than in noVG and mock-infected cells, respectively (mean expression: VG=0.394, noVG=0.262, mock=0.237).\n\nA literature search reviewed some known functions of UBP1 (a.k.a. LBP-1a and NF2d9) related to this study:\n- Can form a heteromeric DNA binding complex with cousin proteins TFCP2 and TFCP2L1 (To et al., PLoS One 2010), which TFCP2 could mediate Snail1-induced EMT (Porta-de-la-Riva et al., Biochem J. 2011)\n- UBP1 knockout mice exhibit retarded growth and has a role in angiogenesis and blood pressure (Parekh et al., Mol. Cell Biol. 2004; Koutnikova et al., PLoS Genet. 2009)\n- For HIV-1, UBP1 can repress HIV-1 transcription via c-Myc (Stojanova et al., J Cell Biochem. 2004), or suggested that the UBP1/TFCP2 and UBP1/MED16 complex can bind to the transcription start site of HIV-1 genome to inhibit HIV-1 gene transcription and reinforce latency (Kato et al., Science 1991; Zheng et al., eLife 2025)\n- Looking at the Gene Transcription Regulation Database (GTRD) (a ChIP-seq database; Kolmykov et al., Nucleic Acids Res. 2021), it appears that UBP1 can bind to the promoters of multiple genes. Interestingly, out of the genes we examined in the scRNA-seq data on EMT or TGF- signaling, we found that TGFB1, SMAD3, COL1A1, VIM, RAF1, and NFKB2, could be potential targets of UBP1.\n\nTherefore, UBP1 emerges as a compelling candidate for follow-up investigation. It may represent a host factor upregulated in association with HCMV presence, potentially serving as a link between viral gene expression and the modulation of UBP1-dependent pathways that influence TGF-β signaling and EMT. A critical next step will be to confirm whether UBP1-expressing cells directly harbor HCMV DNA. We emphasize that these data and points are preliminary and are not included in the current manuscript.\n\nIn addition to our initial marker gene investigations, we conducted a comparative analysis of EMT- and TGF-β signaling-related genes between the viral gene-positive (VG) and viral gene-negative (noVG) subpopulations. The results show that expression of EMT-related genes such as CDH2, COL1A1, VIM, and S100A4 is present in both subpopulations, with marginally higher levels (within a 10% difference) observed in the noVG cells (Letter Figure 1A). Similarly, key EMT-inducing transcription factors (SNAI1, TWIST2, ZEB1) were also more highly expressed in the noVG cells (Letter Figure 1B).\n\nInterestingly, this contrasts with the expression of the upstream cytokine TGFB1, which was higher in the VG cells (Letter Figure 1C). Consistent with this, downstream signaling components of the TGF-β pathway—including the canonical SMAD2/3 and the non-canonical MAPK1 pathways—also showed evidence of stronger activation in the VG subpopulation (Letter Figure 1D).\n\nThese preliminary analyses suggest that viral gene expression during latent infection in cholangiocytes may relate to the induction of TGF-β, which could then act in an autocrine or paracrine manner to promote EMT. Interestingly, TGF-β appears to induce a higher level of EMT in cells not expressing viral genes—including non-infected cells within the HCMV-infected organoids. These observations provide a useful starting point for further investigation into the specific viral genes responsible for inducing TGF-β and EMT, as well as for characterizing the cell types that support latent and productive infection. We thank the reviewer for raising this point, as it has allowed us to extend our analysis and gain deeper insight into the interaction between HCMV and cholangiocyte organoids. The data shared here will not be included in the revised manuscript.\n\nAuthors: Thank you for the comment. In Supplementary Figure S4, we have revised the panels to provide a clearer view of the organoid cultures. Panel A now shows a larger, higher-resolution overview of the organoids. Panel B displays zoomed-in images of individual organoids. The regions of interest (ROIs) used to measure fluorescence intensity for CK19 (Suppl. Fig. 4a) and Sox9 (Suppl. Fig. 4b) in each organoid are clearly indicated: a white outline denotes non-GFP+ organoids (Inf.), while a cyan outline denotes GFP+ organoids (Inf. (GFP+)). The corresponding intensity values for individual organoids are graphed to the right of each image set, with the mean and standard deviation displayed. Statistical analysis was performed using a one-way ANOVA. We believe these revisions provide a clearer and more precise presentation of the data.\n\nAuthors: Thank you for highlighting this discrepancy. We have addressed the imbalance in organoid counts by analyzing an additional 18 organoids in the mock condition, resulting in comparable sample sizes (mock: n=44, HCMV: n=44). A one-way ANOVA comparing timepoints within the mock culture confirms a significant increase in organoid size over time, a trend not observed in the HCMV-infected culture. Furthermore, direct comparisons between mock and infected organoids at each timepoint revealed a significant decrease in size due to infection. These corrected analyses are now presented in the revised Figure 3b, where values are shown as the percentage of change compared to the day 0 PI.\n\nAuthors: Thank you for bringing this to our attention. We sincerely apologize for the error introduced during the revision process; the y-axis labels were inadvertently reversed. The correct order, from top to bottom, should be 5, 4, 3, 2, 1. This correction ensures the figure now matches the data presented in Figure 5c and is consistent with the corresponding text in the manuscript. We regret any confusion this may have caused.\n\nAuthors: We agree that the original magnification was insufficient to clearly visualize the nuclear HCMV-probe signal. In response, we have revised Figure 6b to include larger images and zoomed-in insets for both cholangiocytes and hepatocytes, contrasted with HCMV-probe negative control samples of both cell types. The panel has been rearranged to ensure all images are presented at a sufficient scale for clear interpretation.\n\nAuthors: Thank you for your thoughtful comments and helpful suggestions. We sincerely appreciate the time and effort you have dedicated to reviewing our work and providing such constructive feedback."}, {"round": 3, "reviewer_comments": {"reviewer_3": "This reviewer appreciates all the additional work the authors have performed and look forward to future studies showing a more \"granular\" analysis of the gfp- population of cells within their infected organoids. However, this reviewer is afraid that the authors have misinterpreted my comments regarding the presentation of their data. They certainly have acknowledged that there are gfp+ and gfp- cells within their infected organoids. However, what my suggestion entailed was to show three groups of organoid/cells: 1) mock infected 2) infected (which would contain ALL infected cells, regardless of gfp status) and 3) gfp+ infected cells (which are, as we know, a minor population).\nAll the authors have done is to \"rename\" their gfp- subpopulation as \"infected\". This does not address my main point/suggestion, which was to treat the infected organoids EITHER as an entire grouping (gfp+ and gfp- cells altogether) or to separate out and show information from the subgroup of cells that are CLEARLY infected and expressing viral proteins and shedding virus.\nIf the authors are not going to take my suggestion, I would encourage them to NOT call the subgroup of gfp- cells \"INFECTED\" - this is a misnomer, as it does not include the group of cells that ARE CLEARLY infected (and gfp+).\nAlong this line, in the first introduction of your groups on lines 139-143, you call the two groups \"gfp+\" and \"non-gfp-\" populations. Those would be the same thing….. I think you meant to say \"non-gfp+\". In which case, once again, I would revert back to gfp-.\nAll the other changes you have made to figures to address my other comments are fine and clarify my concerns."}, "author_response": "Authors: We apologise for misinterpreting this reviewer’s comments regarding the suggestion for the three groups, and we appreciate the reviewer’s clarifications. Understandably, in the initial submission, other reviewers raised concerns to not distinguish between the GFP- and GFP+ subpopulation of cells in the HCMV-infected organoids. Thus, in the subsequent manuscript, we performed vast experiments to demonstrate the differences of the two and presented as such. If we are to show the total cells in infected organoids and then the gfp+ cells in the organoids, we believe we will be showing similar data to the initial version of the manuscript. We think that the data presented now as HCMV-GFP- and HCMV-GFP+ cells has a better comparison of the similarity and differences in the behaviour of the virus infection, EMT, and TGF-beta signalling. Therefore, we will keep the current analysis.\n\nAuthors: The non-gfp+ labels have been reverted to gfp-.\n\nAuthors: We thank and appreciate the reviewer for the constructive comments."}]

Decision letter