Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in

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University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
James Van Etten Publications Plant Pathology Department
11-2014
Chlorovirus ATCV-1 is part of the human
oropharyngeal virome and is associated with
changes in cognitive functions in humans and mice
Robert H. Yolken
Johns Hopkins School of Medicine, [email protected]
Lorraine Jones-Brando
Johns Hopkins School of Medicine
David D. Dunigan
University of Nebraska-Lincoln, [email protected]
Geetha Kannan
Johns Hopkins School of Medicine
Faith Dickerson
Sheppard Pratt Health System
See next page for additional authors
Follow this and additional works at:http://digitalcommons.unl.edu/vanetten
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Yolken, Robert H.; Jones-Brando, Lorraine; Dunigan, David D.; Kannan, Geetha; Dickerson, Faith; Severance, Emily; Sabunciyan,
Sarven; Talbot, C. Conover Jr.; Prandovszky, Emese; Gurnon, James R.; Agarkova, Irina V.; Leister, Flora; Gressitt, Kristin L.; Chen,
Ou; Deuber, Bryan; Ma, Fangrui; Pletnikov, Mikhail V.; and Van Etten, James L., "Chlorovirus ATCV-1 is part of the human
oropharyngeal virome and is associated with changes in cognitive functions in humans and mice" (2014).James Van Etten Publications.
8.
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Authors
Robert H. Yolken, Lorraine Jones-Brando, David D. Dunigan, Geetha Kannan, Faith Dickerson, Emily
Severance, Sarven Sabunciyan, C. Conover Talbot Jr., Emese Prandovszky, James R. Gurnon, Irina V.
Agarkova, Flora Leister, Kristin L. Gressitt, Ou Chen, Bryan Deuber, Fangrui Ma, Mikhail V. Pletnikov, and
James L. Van Etten
This article is available at DigitalCommons@University of Nebraska - Lincoln:http://digitalcommons.unl.edu/vanetten/8
Chlorovirus ATCV-1 is part of the human oropharyngeal
virome and is associated with changes in cognitive
functions in humans and mice
RobertH.Yolkena,1,LorraineJones-Brandoa,DavidD.Duniganb,GeethaKannanc,FaithDickersond,EmilySeverancea,
SarvenSabunciyana,C.ConoverTalbotJr.e,EmesePrandovszkya,JamesR.Gurnonb,IrinaV.Agarkovab,FloraLeistera,
KristinL.Gressitta,OuChena,BryanDeubera,FangruiMab,MikhailV. Pletnikovc,andJamesL.VanEttenb,1
aStanleyDivisionofDevelopmentalNeurovirology,DepartmentofPediatrics,cDepartmentofPsychiatryandBehavioralSciences,andeInstituteforBasic
BiomedicalSciences,JohnsHopkinsSchoolofMedicine,Baltimore,MD21205;bNebraskaCenterforVirologyandDepartmentofPlantPathology,University
ofNebraska,Lincoln,NE68583-0900;anddDepartmentofPsychology,SheppardPrattHealthSystem,Baltimore,MD21205
ContributedbyJamesL.VanEtten,October3,2014(sentforreviewAugust9,2014;reviewedbyJoramFeldonandAllanV.Kalueff)
Chloroviruses (family Phycodnaviridae) are large DNA viruses In the process of analyzing whole genome sequences obtained
knowntoinfectcertaineukaryoticgreenalgaeandhavenotbeen fromunfractionatedsamplesoftheoropharynxfromhealthyindi-
previously shown to infect humans or to be part of the human viduals participating in a study that involved the assessment of
virome. We unexpectedly found sequences homologous to the cognitive functioning, we unexpectedly discovered a substantial
chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in numberofsequencereadsverysimilartovirusAcanthocystisturfa-
ametagenomicanalysisofDNAextractedfromhumanoropharyn- ceachlorellavirus1(ATCV-1),amemberofthegenusChlorovirus
geal samples. These samples were obtained by throat swabs of
(family Phycodnaviridae). This family of algae-infecting viruses is
adults without a psychiatric disorder or serious physical illness
common in aqueous environments but not previously thought to
whowereparticipatinginastudythatincludedmeasuresofcog-
nitivefunctioning.ThepresenceofATCV-1DNAwasconfirmedby infect humans or animals or to inhabit human mucosal surfaces
quantitativePCRwithATCV-1DNAbeingdocumentedinoropha- (13). Viruses that cross kingdoms are rare; however, some plant
ryngealsamplesobtainedfrom40(43.5%)of92individuals.The viruses can replicate in both their plant host as well as an in-
presence of ATCV-1 DNA was not associated with demographic vertebratevector.However,thereisonereportindicatingapossible
variablesbutwasassociatedwithamodestbutstatisticallysignif- algal-infectingvirusassociatedwithhumans.Inthisreport,cervico-
icantdecreaseintheperformanceoncognitiveassessmentsofvisual vaginal secretion samples contained virus-like particles, and these
processingandvisualmotorspeed.Wefurtherexploredtheeffectsof
samplesinhibitedthepropagationofcertainalgalcultures,consis-
ATCV-1inamousemodel.TheinoculationofATCV-1intotheintes-
tentwiththepresenceofaviruscapableofinfectingalgae(14).
tinaltractof9–11-wk-oldmiceresultedinasubsequentdecreasein
performance in several cognitive domains, including ones involving
Significance
recognitionmemoryandsensory-motorgating.ATCV-1exposurein
micealsoresultedinthealteredexpressionofgeneswithinthehip-
pocampus.Thesegenescomprisedpathwaysrelatedtosynapticplas- Humanmucosalsurfacescontainawiderangeofmicroorganisms.
ticity, learning, memory formation, and the immune response to The biological effects of these organisms are largely unknown.
viralexposure. Large-scale metagenomic sequencing is emerging as a method
to identify novel microbes. Unexpectedly, we identified DNA
| | |
chlorovirusATCV-1 infection cognitivefunctioning sequences homologous to virus ATCV-1, an algal virus not
|
oropharyngealvirome metagenomicsequencing previously known to infect humans, in oropharyngeal samples
obtainedfromhealthyadults.ThepresenceofATCV-1wasasso-
Mucosal sites of humans such as the oral pharynx and in- ciatedwithamodestbutmeasurabledecreaseincognitivefunc-
tioning.ArelationshipbetweenATCV-1andcognitivefunctioning
testinal tract are not sterile but contain many micro-
wasconfirmedinamousemodel,whichalsoindicatedthatex-
organisms including bacteria, viruses, and fungi. It has recently
posuretoATCV-1resultedinchangesingeneexpressionwithin
become apparent that individuals differ in the composition of the
microbialfloraattheirmucosalsites,characterizedasthe“micro- thebrain.Ourstudyindicatesthatvirusesintheenvironmentnot
biota” comprising the “microbiome.” Studies in both humans and thoughttoinfecthumanscanhavebiologicaleffects.
animal models have shown that the microbiome can affect bio-
logical functions, including cognitive performance (1, 2). Bacterial Authorcontributions:R.H.Y.,L.J.-B.,M.V.P.,andJ.L.V.E.designedresearch;L.J.-B.,G.K.,
F.D.,E.S.,S.S.,J.R.G.,I.V.A.,F.L.,K.L.G.,O.C.,B.D.,andM.V.P.performedresearch;R.H.Y.
and fungal components of the oral microbiome have been the andL.J.-B.supervisedtheoverallperformanceoftheanalyses;D.D.D.andF.M.mapped
subject of several prior studies. These studies generally rely on thevirusgenes;G.K.performedtheanimalinfectionandbehaviorexperiments;F.D.
PCR-based amplification and subsequent analysis of ribosomal supervisedthecollectionofthehumansamples;E.S.supervisedtheprocessingofthe
RNA sequences conserved among most species (e.g., refs. 3–6). humansamplesandthemeasurementoftheantibodiesinthemousesamples;S.S.su-
pervisedtheexperimentsrelatedtohighthroughputsequencing;C.C.T.andE.P.per-
Analysis of viral components in the microbiome (virome) has re- formedtheanalysisofgeneexpression;J.R.G.testedforinfectiousvirusandproduced
ceived less attention, largely because there are no universal target thevirus;I.V.A.testedforinfectiousvirus;F.L.performedtheexperimentsrelatedtoviral
regions suitable for PCR amplification across different viral taxa. DNAdetection;K.L.G.performedtheantibodymeasurementstudies;O.C.performedthe
experimentsrelatedtohighthroughputsequencing;B.D.producedthevirus;M.V.P.
This problem can be overcome by whole genome sequencing in
supervisedtheanimalinfectionandbehaviorexperiments;D.D.D.,C.C.T.,E.P.,andF.M.
whichviralparticlesareseparatedfromtherestofthesample,se- analyzeddata;andR.H.Y.,L.J.-B.,D.D.D.,M.V.P.,andJ.L.V.E.wrotethepaper.
quenced,andthenbioinformaticallyidentified.Suchanalyseshave
Reviewers:J.F.,TheSwissFederalInstituteofTechnology;andA.V.K.,TulaneUniversity.
identifiedbacteriophagesasthe most abundant component of the
Theauthorsdeclarenoconflictofinterest.
humanoralmicrobiome(6–9).Theviromecanalsobeevaluatedby
1Towhomcorrespondencemaybeaddressed.Email:[email protected]@
sequencing unfractionated samples. This approach has the advan-
gmail.com.
tage of identifying viruses that might be in low concentrations or
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
evadedetectionbystandardfractionationmethods(10–12). 1073/pnas.1418895111/-/DCSupplemental.
16106–16111 | PNAS | November11,2014 | vol.111 | no.45 www.pnas.org/cgi/doi/10.1073/pnas.1418895111
The surprising discovery of this apparent human–ATCV-1
association led us to conduct further investigations in humans
andinmicebyusinghighthroughputsequencingmethods,PCR
procedures,andimmunologicalanalyses.ThepresenceofATCV-1
genomesintheoropharynxofmanyindividualswasevaluatedin
a Baltimore, Maryland-based cohort, and correlations were dis-
covered between the presence of ATCV-1 and cognitive per-
formance. These results prompted us to explore the ability of
ATCV-1 to infect mice under experimental conditions and to
study the effect of ATCV-1 infection on cognitive performance
andbraingene expression.
Results
SAG 3.83_ATCV-1
Detection of ATCV-1 DNA in Human Pharyngeal Samples. Meta-
genomic sequencing was performed on DNA extracted from oro-
pharyngeal samples obtained from 33 adult individuals without
a known psychiatric disorder or physical illness. The demographic
characteristicsoftheseindividualsarereportedinTableS1A.The
viralfractionofthesemetagenomicanalysesrevealedawiderange
of virusesconsistent with human and bacterial components of the
oropharyngeal cavity. However, there were an unexpected signifi-
cant number of sequences that resembled chlorovirus ATCV-1.
IndividualswithandwithoutdetectableATCV-1sequencesintheir
oropharyngeal samples did not differ significantly in terms of the
demographicvariablesof age, sex, race, educational level, level of
maternaleducation,cigarettesmoking,basalmetabolicindex(BMI)
score,ahistoryoftraveloutsideofNorthAmerica,orplaceofbirth. Fig.1. ChlorovirusATCV-1genomeshowingthegeneblockdistributions
The sequence reads mapped to many ATCV-1 sites located [blue arrows, protein coding sequence (CDS); red arrows, tRNAs] on each
strand of the genome. Histograms in black indicate the G+C distribution
throughouttheviral genome(Fig.1andTableS2).
along the genome; colored histograms (green, magenta) indicate the GC
A quantitative PCR (qPCR) procedure with a fluorescent-
skewofthegenome.Themostinnercircleindicatesthegenomemappo-
labeledprobe(Taqman)wasdevelopedtoallowthroatsofmore sitionwiththestartpositionat“12o’clock.”Theviralgenomeisalinear
individuals to be tested for ATCV-1 DNA. The assay relied on dsDNA,butisrepresentedhereasacircleforconvenienceofpresentation.
primers directed at ATCV-1 gene z100l. The sensitivity of the Control throat swab deep sequencing consensus sequence reads are
assay was ∼10 copies of target DNA based on standard curves matched to ATCV-1, and two experiments (17 and 16 individuals per ex-
generated from purified ATCV-1 DNA. The qPCR assay periment) arerepresentedbytheblacklines connectingthegene blocks.
detected ATCV-1 DNA in all 10 individuals with at least two BLAST hits, 61; Query, ATCV-1; Subject, human throat swab chlorovirus
sequence reads homologous to ATCV-1 and in 12 of the 14 consensussequencereads(52).
individualswhohadonesequencereadhomologoustoATCV-1.
TheTaqmanassaywasnegativewhentestedwitheitherhuman
the Wechsler Adult Intelligence Scale (WAIS) Information
DNA or extracts from buffer solutions. Furthermore, the assay
subtest,atestofgeneralknowledge.
wasnegative,withDNAextractedfromeithertheATCV-1host
Theoddsratiosdefiningtheassociationbetweenthepresence
Chlorella heliozoae or with DNA from two other chloroviruses,
of oropharyngeal ATCV-1 DNA and low performance on the
PBCV-1 and CVM-1, and their hosts Chlorella variabilis and
Micractiniumconductrix,respectively. cognitivetestsweresignificantlycorrelated.AsdepictedinFig.2,
thepresenceofATCV-1oropharyngealDNAwasassociatedwith
The Taqman assay was used on oropharyngeal samples from
low performance on Trails A with an odds ratio of 5.2 (95%
92individuals,includingthe33individualstestedabove.Overall
ATCV-1–like DNA was detected in 40 (43.5%) of the 92 sam- confidence interval, 1.63–16.7; P < 0.005) and low performance
on the RBANS Total Score with an odds ratio of 4.3 (95% Y
ples.IndividualswithorwithoutdetectableATCV-1DNAwere G
similar in terms of demographic variables (Table S1B). No confidenceinterval,1.4–12.8;P<0.01).WithintheRBANSthere OLO
ATCV-1DNAwasdetectedinbloodsamplesobtainedfromthe was a strong association between oropharyngeal ATCV-1 DNA OBI
stuBdeycianudsievitdhuealisndbiyvitdhueaqlsPiCnRthaesssatuy.dy cohort were also partici- a9n5%d lcoownfpideerfnocremianntecrevaoln,1t.h7e–3a7t.t6e;nPtio<n0d.0o0m8)a.inTh(eosdedassrsoatciioa,ti8o.n0s; MICR
patinginastudyofcognitivefunctioning(15),weexaminedthe were independent of the covariates of age, sex, race, socioeco-
association between detection of ATCV-1 DNA and perfor- nomic status, educational level, place of birth, and current ciga-
mance on a battery of cognitive tests. A significant association rettesmoking.Nosignificantdifferencesoccurredwithalowlevel
occurredbetweenthepresenceoforopharyngealATCV-1DNA ofperformance onthe other RBANS domains or on thetest of
andalowerlevelofperformanceontheTrailMakingTestPart information(allP>0.1).
A(TrailsA),atestofvisualmotorspeed(P<0.002),aswellas
thetotalscoreoftheRepeatableBatteryfortheAssessmentof EffectofATCV-1onMouseBehaviorandCognition.A series of be-
Neuropsychological Status (RBANS) (P < 0.014) (Table 1). havior tests were performed on an equal number of male and
Within the RBANS test, there were statistically significant dif- femalemicegavagedwitheitherC.heliozoaealone(control,n=
ferences between those who had detectable oropharyngeal 20) or C. heliozoae infected with ATCV-1 at a multiplicity of
ATCV-1DNAandthosewhodidnotinthedomainsofdelayed infection of 10 per cell for 5 h (C. heliozoae/ATCV-1 exposed,
memory(P<0.039)andattention(P<0.011).Thesedifferences n = 30). The behavior tests were started 6 wk postinoculation.
were independent of the covariates of age, sex, race, socioeco- Anopenfieldtestanddark–lightboxwereusedtoevaluatethe
nomic status, educational level, place of birth, and current cig- effects of viral exposure on general locomotor activity and anx-
arettesmoking.Ontheotherhand,nodifferenceswereobserved iety(16,17).Nosignificantgroupdifferencesoccurredineither
between the presence/absence of ATCV-1 DNA and scores on test. The effect of ATCV-1 exposure on learning and memory
Yolkenetal. PNAS | November11,2014 | vol.111 | no.45 | 16107
Table1. AssociationbetweenATCV-1oropharyngealDNAandperformanceoncognitivetests
CognitiveTest ATCV-1DNAdetected,n=40 ATCV-1DNAnotdetected,n=52 Overallcohort,n=93 Pvalue
TrailsA,scaledscore 38.2(12.4) 46.7(11.7) 43.0(12.7) <0.002
WAISIII,Informationsubtest,scaledscore 10.8(2.7) 10.8(2.6) 10.8(2.6) NS
RBANS
TotalScore 81.3(11.9) 85.4(11.5) 83.6(11.8) <0.014
AttentionIndex 91.4(17.5) 98.5(14.5) 95.4(16.2) <0.011
DelayedMemoryIndex 85.2(11.7) 88.3(9.9) 87.0(10.8) <0.039
ImmediateMemoryIndex 85.8(15.5) 89.3(14.5) 87.8(14.9) NS
Visuospatial/ConstructionalIndex 72.6(9.1) 74.4(10.6) 73.6(9.9) NS
LanguageIndex 93.3(17.0) 94.8(17.0) 94.2(16.9) NS
Valueslistedaremeans(standarddeviations).Pvaluescalculatedbylinearregressionadjustedforage,sex,race,educationallevel,maternaleducation,
cigarettesmoking,andplaceofbirth.NSindicatesP>0.1.
was then tested using the Y-maze test to evaluate spatial rec- AntibodiestoATCV-1inExposedMice.Followingthecompletionof
ognitionmemory(17,18).MiceexposedtoATCV-1performed thebehavioralstudies,therewereatotalof47micefromwhich
atalowerlevelonthistestcomparedwithcontrolmice(Fig.3A). serum could be obtained for antibody testing ∼6 mo following
ANOVAofthepercentageofentriestothepreviouslyblockedarm oral inoculation. This set included 28 of the 30 mice that had
showedasignificanteffectofgroup,F(1,49)=6.4,P=0.015.This beeninoculatedwithATCV-1–infectedC.heliozoae(15females,
difference could not be explained by lower locomotor activity in 13 males; 2 males died before testing) and 19 mice inoculated
ATCV-1–exposedmice,asbothgroupshadsimilarnumbersoftotal with C. heliozoae alone (10 males, 9 females; 1 female died be-
entrancesovertheobservationalperiod:19.8±1.45forthecontrol fore testing). We found detectable antibodies to ATCV-1 by
group and 19.5 ± 1.39 for the ATCV-1 group. The effects of enzyme immunoassay (ELISA) in 10 of the 28 mice exposed to
ATCV-1 exposure on recognition of a novel object or a novel lo- ATCV-1 (5 males and 5 females) but in none of the 19 mice
cationwerethenevaluated.Thesetestsevaluatedifferentaspectsof exposedtoC.heliozoae alone(P<0.0033,Fisher’sexacttest).
recognitionmemoryinmice(19).Nodifferenceswereobservedin ThepresenceofantibodiestoATCV-1proteinswasexaminedby
baselineexploratoryactivityoftheobjectsduringtrainingbetween Western blot in 12 available blood samples from mice exposed to
the ATCV-1–exposed and control group. In contrast, compared ATCV-1inthisstudy.Offivetestedsamplesthatwerepositiveby
with control mice, ATCV-1–exposed mice spent significantly less ELISA,fouralsoreactedtomultipleATCV-1proteinsbutnotto
timeexploringthenovelobject,F(1,48)=62.3,P<0.001(Fig.3B), C. heliozoae proteins (Fig. S9). One of the predominant proteins
orthenovellocationofthesameobject,F(1,47)=7.75,P=0.008 recognized was tentatively identified as the ATCV-1 major capsid
(Fig.3C).Thepassiveavoidancetestwasusedtoevaluatememory protein.NoreactiontoATCV-1proteinsoccurredinWesternblots
to aversive stimuli in mice (20). No significant effect of virus ex- with the seven samples that were seronegative with ELISAs. Sera
posurewasobservedinthistest. obtained from mice exposed to C. heliozoae in the absence of
TheeffectsofATCV-1exposureontheacousticstartleandits ATCV-1didnotreactwithATCV-1proteins.
prepulseinhibition (PPI)wereassessed.Thesearetranslational
Discussion
measures of sensorimotor gating that are impaired in patients
with neurological and psychiatric illnesses (21). No group dif- Metagenomic sequencing of DNA extracted from human oro-
ferencewasfoundinthebaselineacousticstartleresponse,and pharyngeal samples identified sequences homologous to chlor-
both groups exhibited comparable amplitudes (in mV): 193.1 ± ovirus ATCV-1 in 14 of 33 (42.4%) individuals from a cohort of
8.4forcontrolmiceand212.4±8.6forATCV-1–exposedmice. adults without a known psychiatric disorder or physical illness
However, ATCV-1 exposure impaired PPI in mice. Two-way who were living in an urban area in the United States. DNA
repeatedmeasuresANOVAwithtreatmentasabetween-subject sequences found in study individuals mapped to many sites on
factor and PPI as a within-subjects factor indicated a significant theATCV-1genomeandincludedmanyviralgenes(Fig.1).The
groupeffect,F(1,45)=6.9,P=0.015,andasignificanteffectoftype finding of ATCV-1 sequences by metagenomic sequencing was
of prepulse, F(4, 234) = 98.2, P < 0.001 (Fig. 3D). No Group × confirmed by a sequence-specific (gene z100l) qPCR assay that
Prepulse interaction was found. Post hoc test results revealed that detected ATCV-1 sequences in oropharyngeal samples from 40
compared with control micethe ATCV-exposed grouphad signifi- of 92 (43.5%) individuals from the same study cohort. There
cantlyreducedPPIaveragedacrossallprepulses(P<0.05)(Fig.3E). werenodifferencesbetweenindividuals,withorwithoutATCV-1
sequences, with respect to demographic variables, such as age,
Hippocampus Gene Expression in ATCV-1–Exposed Mice. Gene ex- sex,race,socioeconomicstatus,cigarettesmoking,travelhistory,
pression in the hippocampus of mice was evaluated to compare orplaceofbirth.
ATCV-1 exposure (26 wk after ATCV-1 exposure) and control Asnotedintheintroduction,ATCV-1isamemberofthegenus
mice.Thehippocampuswasselectedbecauseitcontainspathways Chlorovirus, family Phycodnaviridae. Viruses in the phycodnavirus
essentialforlearning,memory,andbehavior(22,23).Exposureto family, together with those in the Poxviridae, Iridoviridae, Ascovir-
ATCV-1 was associated with a significant up-regulation or down- idae,Asfarviridae,Mimiviridae,andMarseilleviridae,areproposedto
regulation of 1,285 individual genes (Fig. S1), which could be haveacommonevolutionaryancestorandareoftenreferredtoas
constructed into 34 functional pathways that met the inclusion nucleocytoplasmic large DNA viruses (24–26). Chloroviruses have
criteria(TableS3).Pathwayswithmultiplecomponentsrelevantto largedsDNAgenomes(290–370kb)thatencodeupto410proteins
theresponseofthemicetoATCV-1exposureandthedevelopment and many tRNAs (13). Chloroviruses infect certain unicellular,
ofcognitivechangesfollowinginfectionincludepathwaysrelatedto eukaryotic, exsymbiotic chlorella-like green algae, called zoochlor-
dopamine receptor signaling (Fig. S2), cyclin-dependent kinase 5 ellae.ZoochlorellaeareassociatedwiththeprotozoanParamecium
(CDK5)signaling(Fig.S3),antigenpresentation(Fig.S4),immune bursaria, the coelenterate Hydra viridis, the heliozoon A. turfacea,
cell adhesion (Figs. S5 and S6), and eukaryotic initiation factor 2 and other freshwater and marine invertebrates and protozoans
(EIF2)(Fig.S7)withcolorcodinglegend(Fig.S8). (27, 28). Three such zoochlorellae are Chlorella NC64A (renamed
16108 | www.pnas.org/cgi/doi/10.1073/pnas.1418895111 Yolkenetal.
RBANSAttention,bothofwhichmeasurevisualprocessingand
visualmotorspeed.
The association between the level of ATCV-1 and decreased
performanceoncognitivetestswasindependentofdemographic
variables (Tables S1A and S1B). Also, there was no association
between the level of oropharyngeal ATCV-1 DNA and WAIS
Information, indicating that the association between ATCV-1
DNA and the other cognitive domains was not due to level of
general knowledge or educational background. Studies of asso-
ciationsbetweenenvironmentalfactorsandcognitiveperformance
in humans must always be interpreted with caution because it is
possiblethatexposuretoasingleinfectiousagentmightbeassoci-
ated with unmeasured exposures, such as other infectious agents,
heavy metals, pollutants, or other environmental factors that also
canbeassociatedwithalterationsincognitivefunctioning(33).
For this reason, we developed a mouse model for assessing
behavior and cognitive functioning following ATCV-1 exposure
Fig.2. OddsofdetectingATCV-1inthepharynxbypercentileofscoreon bytheoralroute.Asagroup,miceinoculatedbytheoralroute
cognitivetesting.Barsrepresentthemeanand95%confidenceintervalodds showed signs of impairment in new object or new location rec-
ofdetectingATCV-1DNAintheoropharynxinindividualswiththeindicated
ognitionmemoryandsensory-motor gating asmeasuredbyPPI
test.Theoddsratiosareadjustedforthedemographicvariablesofage,sex,
severalmonthsafter asingleviral exposure(Fig.3).
race, maternal education, educational status, and place of birth in the
ExposureofmicetoATCV-1alsoresultedinchangesingene
UnitedStates.TrailsAandInformationareseparatetestsandnotpartof
theRBANS.**P<0.005,*P<0.01,adjustedforthesamecovariates. expressionwithinthehippocampus,theregionofthebrainmost
associated with spatial memory and navigation (22, 34, 35). Of
particularinterestinlightofthecognitiveassayswerealterations
C. variabilis), Chlorella Pbi (renamed M. conductrix), and C. helio- intheCdk5pathway(Fig.S3)becausethispathwayiscentralto
zoae,thehostforATCV-1.ATCV-1isarepresentativeofagroupof learning and memory formation (36). Also of note were differ-
viruses that infect C. heliozoae and collectively are referred to as encesinexpressionofgenesinthedopaminepathway(Fig.S2)
SAGviruses. because perturbation of the dopamine system impairs novel
Genomesfrom41chlorovirusesinfectingthesethreehostshave objectrecognitionandPPIinrodents(37,38),asdoalterations
beensequenced,assembled,andannotated(29).Collectively,the41 in the pathway of EIF2 because this factor is central to the
virusesencodegenesfrom632proteinfamilies,whereasanygiven controloflong-termsynapticplasticityandmemorystorage(39).
virusonlyhas330–410protein-encodinggenes.Thus,thegenomic Althoughitisdifficulttodirectlyrelatethecognitivetestsused
diversity among these viruses is large. Furthermore, the viruses inourhumanstudywithmemorytestsinmice,itisstrikingthat
encodesomeunusualproteinsthatmightinfluencebrainfunction
includingpotassiumionandaquaglyceroporinchannels,potassium
andcalciumtransporters,polyaminemetabolicenzymes,ahistone
A B C
methyltransferase, and numerous sugar enzymes including several
gw(teyPlloyplFCacrieoclUhdaslsywlol)wlyrthroipeat1vhneni–rrs1tuftieh0mstr0eeeairyslsslPeiaalsFairr.tsUeeerhsic/niomogmhftLhmi.aensiTodrnihtghseeyionnmuvoisbiruaniuosnlsatdeiwnscsdaocptefawhrnaap,nstleeaaor,lqttsuhaieonnth-ufdfergoocwhrtumegztihionhtoeogaurcvsthuelntaonhirrtoees- % Novel Arm Entry12345000000 * % Time with Novel Object12345678000000000 * % Time Loca(cid:415)on at Novel 468200000 *
evidence that the zoochlorellae grow free of their hosts in
D E
indigenouswaters.
100 100
To our knowledge, this is the first report of chlorovirus gene Y
sequencesintheoralpharynxofhumans.Therehavebeenseveral 80 80 * LOG
rweecreentnoretpmoretnstioofnoerda.lTvhireormeeasr,ebtuwtoAeTxCplVan-1atoiornosthfoerrtchhilsoaropvpiaruresenst % PPI 4600 % PPI 4600 ROBIO
C
absence. First, in one report the authors specifically looked for 20 20 MI
knownhumanviralpathogens(30),andsotheywouldhavemissed 0 0
p4 p8 p12 p16 p20
thechloroviruses.Second,inthreeotherreports,thesampleswere Pre-pulse(dB)
filteredthroughboth0.45-μmand0.2-μmfilters;thematerialthat
passed through both filters was used to extract DNA. These Fig.3. BehavioraleffectsoforalATCV-1exposure.Micewereorallyinfec-
researcherswereprimarilyevaluatingbacteriophages(7,8,31).The tedwithC.heliozoaealone(openbars)orwithATCV-1–infectedC.helio-
zoae(solidbars)asdescribedinthetext.(A)Spatialrecognitionmemory;the
icosahedral-shapedchlorovirusesare190nmindiameterandhave
y axis displays the percentage of the previously blocked (i.e., novel) arm
a 34-nm spike structure protruding from one unique vertex (32). entries;*P=0.015measuredbyone-wayANOVA.(B)Novelobjectrecog-
Therefore,itislikelythatATCV-1andmostchloroviruseswouldbe nition;theyaxisdepictsthepercentageoftimespentexploringthenovel
trappedona0.2-μmfilter. object;*P<0.001measuredbyone-wayANOVA.(C)Placerecognitionmemory
The fact that the individuals in our study population were recognition;theyaxisdepictsthepercentageoftimespentexploringthenew
participating in a project, which included measures of cognitive locationofthefamiliarobject;*P<0.008measuredbyone-wayANOVA.(D)
functioning,allowedustoexaminetheassociationofdetectable ImpairedPPI;micewereexposedtopresentationofpulsealone(120dB)and
prepulse–pulsecombinationsacrossdifferentprepulseintensities;forexample,
ATCV-1 DNA in the pharynx and performance on a range of
p4indicatespairingoftheprepulse(4dBabovethebackgroundnoiseof70dB)
cognitive tests. Surprisingly, the presence of ATCV-1 DNA in
withthepulsealone(120dB)(seethetextformoredetails);theyaxisdisplays
the oropharynx was associated with modest but statistically sig- the percentage of PPI. (E) Impaired average PPI; the y axisdisplays the per-
nificantdecreasesinperformanceontestsincludingTrailsAand centageofPPI;*P<0.015measuredbyposthoctest.
Yolkenetal. PNAS | November11,2014 | vol.111 | no.45 | 16109
exposure to ATCV-1 in both humans and mice was associated includingage,self-reportedrace,levelofhighesteducationalattainment,level
withdecrementsinperformanceontaskscallingforvisualspatial ofmaternaleducation,andcurrentuseofcigaretteswereobtainedfromall
abilities (Figs. 2 and 3). More detailed cognitive assessment of participants.Alloftheparticipantsprovidedwritteninformedconsentfollowing
explanationofthestudygoalsandprocedures.Thestudywasapprovedbythe
humansandmiceexposedtoATCV-1mightbetterdefinethese
Institutional Review Boards of Johns Hopkins University and Sheppard
associations and the relationship between exposure to ATCV-1
PrattHospital.
andcognitivefunctioning.
Clinicalsamples.Oropharyngealsampleswereobtainedbyswabbingtheback
There are several questions relating to ATCV-1 exposure in ofthethroatusingsterilecottonswabs(BBLCultureSwabs,BectonDickinson).
humans that remain to be addressed. One concerns the source Onthedayofcollection,theswabsweretransportedfromthecollectionsite
of the acquisition of ATCV-1 in the virome. ATCV-1–like totheprocessinglaboratoryatroomtemperatureandthenfrozenuntil
viruses are common in inland waters such as those around Bal- processedfurther,asdescribedbelow.
timore, so exposure to these water sources would be relatively Cognitivetesting.Alloftheparticipantsunderwentabatteryofcognitivetests,
common. The factors involved in the acquisition of ATCV-1 in aspreviouslydescribed(45).TheseincludedtheRBANS(47),TrailsA(48),and
theInformationsubtestoftheWAISIII(49).Detailsofthesetestsarede-
the oropharynx following exposure will be the subject of addi-
scribedinSIMaterialsandMethods.
tional investigations. ATCV-1 is the reference genome for the
SAG virus group, but we have previously sequenced 12 other Sample Processing. DNA was extracted from throat swabs using Qiagen’s
SAG viruses and there are 2–3 distinct clades (29). Therefore,
GentraPuregeneBuccalCellKit.Thecollectionbrushheadsfromtheswab
theexactnatureofSAGvirusescapableofcolonizingthehuman endswereexcisedandincubatedat65°Covernightinthekitcelllysisso-
oropharynxandothermucosalsitesisalsoanimportantsubject lution. The manufacturer’s instructions were followed with some minor
forfuturestudies. changes to the protocol as follows: (i) During the isopropyl alcohol pre-
Another set of questions concerns the mechanisms by which cipitation,2μLof5mg/mLlinearacrylamide(LifeTechnologies),achemically
ATCV-1 might be associated with alterations in cognitive func- synthesized reagent, was substituted for the glycogen carrier to minimize
possible contamination by reagents extracted from natural sources; (ii) fol-
tioning.Althoughtheexactmechanismsofthebehavioralchanges
lowingthisprecipitationstep,incubationonicewasaddedfor15min;(iii)
intheexposedhostremainunclear,theobservedcognitivedeficits
centrifugationfollowingthe70%(vol/vol)ethanolwashwasextendedto5min
areunlikelytoberelatedtosicknessbehavior,asnoovertsignsof from1min;and(iv)finalelutionofDNAwasreducedfrom100μLto30μL.
malaisewerenotedinexposedmice.Similartoanumberofother
microbialinfections,wethinkthatbothdirectandindirecteffects Metagenomic Sequencing. DNA samples from 33 individuals (two in-
of pathogens could play a role (e.g., refs. 40–42). The finding of dependentexperimentswith17and16individuals)wereanalyzedbymet-
alterations in several pathways involved in antigen processing agenomicsequencing.Thedemographicinformationontheseindividualsis
andimmunecellfunctioninginthehippocampusofmiceexposed reported in Table S1A. The method used for sequencing is detailed in SI
to ATCV-1 (Table S3 and Figs. S4–S6) suggests that immune MaterialsandMethods.
mechanisms maybe involved, ashavebeendocumentedinother
biologicalsystems(43).Wefoundevidenceofanimmuneresponse qPCRAnalysis.Oropharyngealsamplesfromalargersetofindividuals(n=92)
were tested by a qPCR system. These individuals included the 33 individuals
to ATCV-1 in about 35% of mice exposed to ATCV-1 when
evaluated by the initial metagenomic sequencing method. The demographic
measured6mofollowingasingleexposure.Thus,ourserological
informationrelatedtotheseindividualsispresentedinTableS1B.Themethod
andgeneexpressiondataimplicateimmuneresponsetoACTV-1
ofsamplecollectionandDNAextractionwasidenticaltothatusedinthemet-
asamechanismunderlyingthecognitivedeficits.Itisconceivable agenomic sequencing. The qPCR was performed using the 5′–3′ exonuclease
that immune activation produced secretion of proinflammatory activityofThermusaquaticuspolymerase(Taqman)(50).Targetprimerswere
cytokinesthataffectedneuronalfunctioning,leadingtobehavioral directedattheportionofthegenomeencodingATCV-1proteinZ100L.This
abnormalities.Inthiscontext,bothsharedanduniqueprofilesof targetregionwasselectedbecausetheprimersdidnothaveappreciableho-
cytokine up-regulation have been shown for various microbial mologywithanyotherviruses,bacteria,oreukaryoticorganisms.
infections (Borna virus vs. Toxoplasma), and it is plausible that ThemethodusedfortheTaqmanassaywasasfollows:A20×assaymix
wasmadeusingforwardprimer5′-GCAATTCCGATAGTAATGGTCA-3′,
differential neurobehavioral outcomes of different microbial
infections may be at least in part explained by unique “sig- reverseprimer5′-CTTGTTTGGCCTTTCACAAA-3′,andprobe/56-FAM/AG
TAAACCCACACCCTTTGGTAGCCA/36-TAMSp/containing18-μMprimers
natures”ofcytokine expression(44). anda4-μMprobe.A20-μLreactionvolumewasusedusing10μLof2×Gene
Earlier blood samples were not obtained from this cohort of ExpressionMasterMix(AppliedBiosystems)and1μLofthe20×assaymix
micetoavoidaffectingthebehavioraltests.Furtherstudiesofthe withtheremainingvolumeconsistingofinputDNAandsterilewater.The
kinetics of ATCV-1 infection and the immune response to in- qPCRreactionprofileconsistedofonecycleof10minat95°Cfollowedby
fectionarethuswarranted.OurstudiesdocumentthatATCV-1is 45cyclesof15sat95°Cand1minat60°C.Reactionswereperformedin
partofthehumanviromeandisassociatedwithcognitivechanges a Stratagene Mx3005P Thermocycler (Agilent Technology). Results were
in humans and experimentally infected animals. An increased quantified with a standard curve generated from the testing of 10-fold
dilutionsofaplasmidcreatedtocontainthetarget.Testingofthisstandard
understandingoftheroleofATCV-1andrelatedvirusesmaylead
curveindicatedthat10copiesofATCV-1DNAcouldbereliablydetectedin
toanewunderstandingoftheroleoftheoropharyngealviromein theqPCRreaction.Asample,whichcontained≥10copiesofATCV-1geno-
humanhealthandcognition.
micDNA,wasconsideredtocontainATCV-1DNA.DNAextractedfromre-
lated chloroviruses PBCV-1 and CVM-1 (13, 29) and human DNA did not
MaterialsandMethods
produceproductswiththisassay.
StudiesinHumans.
Studypopulation.Thestudycohortconsistedof92individualslivingintheBal- StatisticalAnalysisofViromeStudies.Thedemographiccorrelatesforthe
timore,Marylandmetropolitanareawhodidnothavecurrentorpastpsychiatric presence of ATCV-1 DNA were calculated by χ2 analysis for categorical
disordersandwhodidnothaveaseriousmedicalillnessthatwouldbelikelyto variablessuchassex,race,cigarettesmoking,historyoftraveloutsideof
affect cognitive performance. The overall characterization of the study pop- NorthAmerica,andplaceofbirthandbytwo-wayanalysisofvariancefor
ulation including the methods of recruitment and measures used for their continuousvariablessuch asage,levelofeducation,BMIscore,andma-
characterizationwaspreviouslydescribed(45).Controlindividualswereenrolled ternaleducation,thelatterbeingusedasamarkerofsocioeconomicstatus
aftertheywerescreenedtoruleoutthepresenceofcurrentorpastpsychiatric (51).Individualperformancesonthecognitivetestsdescribedabovewere
disorders with the Structured Clinical Interview for Diagnostic and Statistical furthercomparedwiththeperformanceofindividualswhodidordidnot
ManualofMentalDisorders,FourthEditionAxisIDisorders–NonpatientEdition havedetectableATCV-1genomesintheirpharynxusinglinearregression
(46).Participantsalsometthefollowingcriteria:proficientinEnglish,nohistory modelsincorporatingthecovariatesofage,sex,race,educationallevel,ma-
ofi.v.substanceabuse,absenceofmentalretardation,absenceofHIVinfection, ternal education, cigarette smoking, and place of birth. Logistic regression
absenceofaseriousmedicaldisorderthatwouldaffectcognitivefunctioning, modelswereusedtocalculatetheoddsratios,whichdefinetheassociation
and no indication of alcohol or substance use disorder. Demographic data betweenthepresenceofATCV-1DNAinthethroatwithlowperformanceon
16110 | www.pnas.org/cgi/doi/10.1073/pnas.1418895111 Yolkenetal.
thecognitivetestsasdefinedabove,usingthesamecovariatesthatwereused for 5 h (C. heliozoae/ATCV-1), pelleted (3,800 × g × 5 min, 4 °C), and then
inthelinearregressionmodels.Tohaveadequatestatisticalpower,analysesof resuspended in 0.4× PBS. Mice were gavaged with 0.2 mL of either
cognitivefunctioningwereonlyperformedonthelargercohort(n=92),on C. heliozoae/ATCV-1 (n = 30) or C. heliozoae control preparations con-
whomATCV-1DNAwasdetectedbytheqPCRmethoddescribedabove. taining∼4×107cells(n=20),withbothgroupsequallydividedbetween
malesandfemales.
MouseModelofInfection.Animal model studies were performed to de- ThetestsusedtomonitorthebehaviorofthemiceexposedtoATCV-1,as
terminetheeffectofATCV-1exposureviatheoralrouteoncognitionand wellastheproceduresusedforRNA extraction,microarray analysis, data
otherbehaviors.Theconditionsofviralgrowthandmouseinoculation normalization and statistical analysis for microarray transcriptomics,
weredeterminedbyasetofpreliminaryexperiments.Allprotocolswere pathwayanalysis,andmeasurementofantibodiestoATCV-1andrelated
approved by the Animal Care and Use Committee at Johns Hopkins chloroviruses in mouse blood samples are described in SI Materials
University. andMethods.
Atotalof50C57BL/6maleandfemalemice(CharlesRiverLaboratories)
wereusedtoevaluatetheeffectofATCV-1exposureoncognitionand ACKNOWLEDGMENTS.TheauthorsthankFabriceCasseus,JoshuaCrawford,
behavior. Mice were housed five per cage (28.3 cm length × 17.4 cm RossMcFarland,andChunXiaYangfortheirexcellenttechnicalassistance
width×13cmheight)unlessseparatedduetofighting.Animalshadfree with the mice studies. This work was supported by grants from the
accesstofoodandwateratalltimes.Micewereinoculatedat9–11wkof Stanley Medical Research Institute, National Institute of Mental Health
P50SilvioO.ConteCenteratJohnsHopkins(MH-94268),NationalScience
age,asdescribedbelow. Foundation–Experimental Program to Stimulate Competitive Research
Grant EPS-1004094, and a P20-RR15635 grant from the Centers of Bio-
ExposuretoATCV-1.C.heliozoaehostalgaewereeitheruninfected(C.heliozoae medicalResearchExcellenceprogramoftheNationalCenterforResearch
control)orinfectedwithATCV-1atamultiplicityofinfectionof10PFUpercell Resources.
1. Al-AsmakhM,AnuarF,ZadjaliF,RafterJ,PetterssonS(2012)Gutmicrobialcom- 27.PröscholdT,DarienkoT,SilvaPC,ReisserW,KrienitzL(2011)Thesystematicsof
munitiesmodulatingbraindevelopmentandfunction.GutMicrobes3(4):366–373. Zoochlorella revisited employing an integrative approach. Environ Microbiol
2. DavariS,TalaeiSA,AlaeiH,SalamiM(2013)Probioticstreatmentimprovesdiabetes- 13(2):350–364.
induced impairment of synaptic activity and cognitive function: Behavioral and 28.EstebanGF,FenchelT,FinlayBJ(2010)Mixotrophyinciliates.Protist161(5):621–641.
electrophysiologicalproofsformicrobiome-gut-brainaxis.Neuroscience240:287–296. 29.JeanniardA,etal.(2013)Towardsdefiningthechloroviruses:Agenomicjourney
3. CostelloEK,etal.(2009)Bacterialcommunityvariationinhumanbodyhabitatsacross throughagenusoflargeDNAviruses.BMCGenomics14:158.
spaceandtime.Science326(5960):1694–1697. 30.NakamuraS,etal.(2009)Directmetagenomicdetectionofviralpathogensinnasal
4. NasidzeI,LiJ,QuinqueD,TangK,StonekingM(2009)Globaldiversityinthehuman andfecalspecimensusinganunbiasedhigh-throughputsequencingapproach.PLoS
salivarymicrobiome.GenomeRes19(4):636–643. ONE4(1):e4219.
5. SegataN,etal.(2012)Compositionoftheadultdigestivetractbacterialmicrobiome 31.WillnerD,etal.(2011)Metagenomicdetectionofphage-encodedplatelet-binding
basedonsevenmouthsurfaces,tonsils,throatandstoolsamples.GenomeBiol13(6): factorsinthehumanoralcavity.ProcNatlAcadSciUSA108(Suppl1):4547–4553.
R42. 32.CherrierMV,etal.(2009)Anicosahedralalgalvirushasacomplexuniquevertex
6. WadeWG(2013)Theoralmicrobiomeinhealthanddisease.PharmacolRes69(1): decoratedbyaspike.ProcNatlAcadSciUSA106(27):11085–11089.
137–143. 33.LiuJ,LewisG(2014)Environmentaltoxicityandpoorcognitiveoutcomesinchildren
7. PrideDT,etal.(2012)Evidenceofarobustresidentbacteriophagepopulationre- andadults.JEnvironHealth76(6):130–138.
vealedthroughanalysisofthehumansalivaryvirome.ISMEJ6(5):915–926. 34.CabralHO,etal.(2014)Oscillatorydynamicsandplacefieldmapsreflecthippocampal
8. PrideDT,SalzmanJ,RelmanDA(2012)Comparisonsofclusteredregularlyinterspaced ensembleprocessingofsequenceandplacememoryunderNMDAreceptorcontrol.
shortpalindromicrepeatsandviromesinhumansalivarevealbacterialadaptationsto Neuron81(2):402–415.
salivaryviruses.EnvironMicrobiol14(9):2564–2576. 35.LyonL,SaksidaLM,BusseyTJ(2012)Spontaneousobjectrecognitionanditsrelevance
9. Robles-SikisakaR,etal.(2013)Associationbetweenlivingenvironmentandhuman toschizophrenia:Areviewoffindingsfrompharmacological,genetic,lesionand
oralviralecology.ISMEJ7(9):1710–1724. developmentalrodentmodels.Psychopharmacology(Berl)220(4):647–672.
10.WommackKE,etal.(2012)VIROME:Astandardoperatingprocedureforanalysisof 36.ShahK,LahiriDK(2014)Cdk5activityinthebrain—Multiplepathsofregulation.
viralmetagenomesequences.StandGenomicSci6(3):427–439. JCellSci127(Pt11):2391–2400.
11.SolonenkoSA,et al.(2013)Sequencingplatformandlibrary preparationchoices 37.vandenBuuseM(2010)Modelingthepositivesymptomsofschizophreniainge-
impactviralmetagenomes.BMCGenomics14:320. netically modified mice: Pharmacology and methodology aspects. Schizophr Bull
12.WylieKM,WeinstockGM,StorchGA(2013)Viromegenomics:Atoolfordefiningthe 36(2):246–270.
humanvirome.CurrOpinMicrobiol16(4):479–484. 38.ValsamisB,SchmidS(2011)Habituationandprepulseinhibitionofacousticstartlein
13.VanEttenJL,DuniganDD(2012)Chloroviruses:Notyoureverydayplantvirus.Trends rodents.JVisExp55(55):e3446.
PlantSci17(1):1–8. 39.Costa-MattioliM,SonenbergN(2006)Translationalcontroloflong-termsynaptic
14.StepanovaOA,SolovyovaYV,SolovyovAV(2011)Resultsofalgaevirusessearchin plasticityandmemorystoragebyeIF2alpha.CritRevNeurobiol18(1-2):187–195.
humanclinicalmaterial.UkrainicaBioorganicaActa2:53–56. 40.YolkenRH,TorreyEF(1995)Viruses,schizophrenia,andbipolardisorder.ClinMi-
15.DickersonF,etal.(2014)Associationbetweencytomegalovirusantibodylevelsand crobiolRev8(1):131–145.
cognitivefunctioninginnon-elderlyadults.PLoSONE9(5):e95510. 41.PletnikovMV,MoranTH,CarboneKM(2002)Bornadiseasevirusinfectionofthe
16.BourinM,HascoëtM(2003)Themouselight/darkboxtest.EurJPharmacol463(1-3): neonatalrat:Developmentalbraininjurymodelofautismspectrumdisorders.Front
Y
55–65. Biosci7:d593–d607. G
O
17.PletnikovMV,etal.(2008)InducibleexpressionofmutanthumanDISC1inmiceis 42.KannanG,PletnikovMV(2012)Toxoplasmagondiiandcognitivedeficitsinschizo- L
O
associatedwithbrainandbehavioralabnormalitiesreminiscentofschizophrenia.Mol phrenia:Ananimalmodelperspective.SchizophrBull38(6):1155–1161. BI
Psychiatry13(2):173–186,115. 43.KohmanRA,RhodesJS(2013)Neurogenesis,inflammationandbehavior.BrainBehav RO
18.MelnikovaT,etal.(2006)Cycloxygenase-2activitypromotescognitivedeficitsbutnot Immun27(1):22–32. MIC
increasedamyloidburdeninamodelofAlzheimer’sdiseaseinasex-dimorphicpat- 44.StewartAM,etal.(2014)Cytokineandendocrineparametersinmousechronicsocial
tern.Neuroscience141(3):1149–1162. defeat:Implicationsfortranslational‘cross-domain’modelingofstress-relatedbrain
19.AntunesM,BialaG(2012)Thenovelobjectrecognitionmemory:Neurobiology,test disorders.BehavBrainRes,pii:S0166-4328(14)00549-X,10.1016/j.bbr.2014.08.037.
procedure,anditsmodifications.CognProcess13(2):93–110. 45.DickersonF,etal.(2008)Associationbetweencognitivefunctioning,exposureto
20.TayebiMeybodiK,VakiliZarchA,ZarrindastMR,DjahanguiriB(2005)Effectsofultra- HerpesSimplexVirustype1,andtheCOMTVal158Metgeneticpolymorphismin
lowdosesofmorphine,naloxoneandethanolonmorphinestate-dependentmemory adultswithoutapsychiatricdisorder.BrainBehavImmun22(7):1103–1107.
ofpassiveavoidanceinmice.BehavPharmacol16(3):139–145. 46.FirstMB,SpitzerRL,GibbonM,WilliamsJBW(2002)StructuredClinicalInterviewfor
21.SwerdlowNR,BraffDL,TaaidN,GeyerMA(1994)Assessingthevalidityofananimal DSM-IV-TRAxisIDisorders,ResearchVersion,PatientEdition.(SCID-I/P)(Biometrics
modelofdeficientsensorimotorgatinginschizophrenicpatients.ArchGenPsychiatry Research,NewYorkStatePsychiatricInstitute,NewYork).
51(2):139–154. 47.RandolphC(1998)RBANSManual—RepeatableBatteryfortheAssessmentofNeu-
22.ChenG,KingJA,BurgessN,O’KeefeJ(2013)Howvisionandmovementcombinein ropsychologicalStatus(PsychologicalCorporation,SanAntonio,TX).
thehippocampalplacecode.ProcNatlAcadSciUSA110(1):378–383. 48.ReitanRM(1979)ManualforAdministrationofNeuropsychologicalTestBatteriesfor
23.TsienJZ,etal.(2013)Oninitialbrainactivitymappingofepisodicandsemantic AdultsandChildren(ReitanNeuropsychologyLaboratory,Tucson,AZ).
memorycodeinthehippocampus.NeurobiolLearnMem105:200–210. 49.WechslerD(1997)WechslerAdultIntelligenceScaleIII(PsychologicalCorporation,
24.IyerLM,AravindL,KooninEV(2001)Commonoriginoffourdiversefamiliesoflarge SanAntonio,TX).
eukaryoticDNAviruses.JVirol75(23):11720–11734. 50.BergalloM,etal.(2009)RealtimePCRTaqManassaysfordetectionofpolyomaviruses
25.IyerLM,BalajiS,KooninEV,AravindL(2006)Evolutionarygenomicsofnucleo- KIVandWUVinclinicalsamples.JVirolMethods162(1-2):69–74.
cytoplasmiclargeDNAviruses.VirusRes117(1):156–184. 51.Elo IT (2009) Social class differentials in health and mortality: Patterns and ex-
26.YutinN,WolfYI,RaoultD,KooninEV(2009)Eukaryoticlargenucleo-cytoplasmic planations.Comparativeperspective.AnnuRevSociol35:553–572.
DNA viruses: Clusters of orthologous genes and reconstruction of viral genome 52.GrantJR,ArantesAS,StothardP(2012)Comparingthousandsofcirculargenomes
evolution.VirolJ6:223. usingtheCGViewComparisonTool.BMCGenomics13:202.
Yolkenetal. PNAS | November11,2014 | vol.111 | no.45 | 16111
Supporting Information
Supporting Information Corrected February 20, 2015
Yolken et al. 10.1073/pnas.1418895111
SIMaterialsandMethods Novelobjectrecognition.Thenovelobjectrecognitiontestwasused
Thetestsusedtomonitorcognitivebehavior,thebehaviorof toassessrecognitionmemory(8).Briefly,micewerehabituated
themiceexposedtoATCV-1,aswelltheproceduresusedfor for4dtoanemptymousecage(28.3cmlength×17.4cmwidth×
metagenomic sequencing, RNA extraction, microarray analysis, 13cmheight)for10mineachdayaspreviouslydescribed(9,10).
data normalization and statistical analysis for microarray tran- Onday 5, two identical objectswere placed onopposite ends of
scriptomics,pathwayanalysis,andmeasurementofantibodiesto theemptycage,andthemousewasallowedtofreelyexplorethe
ATCV-1andrelatedchlorovirusesinmousebloodsamplesfollow. objectsfor10min.Aftera1-hdelay,duringwhichthemousewas
heldinitshomecage,oneofthetwofamiliarobjectswasreplaced
CognitiveTesting.All of the participants underwent a battery of withanovelone,andthemousewasallowedtofreelyexplorethe
cognitive tests, as previously described (1). These included the familiarandnovelobjectfor5min.Thepercenttimenearthenovel
Repeatable Battery for the Assessment of Neuropsychological objectwascalculatedasthetimenearthenovelobjectdividedby
Status (RBANS) (2), Trail Making Test Part A (Trails A) (3), thetotaltimeneareitherobject.
and the Information subtest of the Wechsler Adult Intelligence Novellocationrecognition.Novellocationrecognitionwasusedto
Scale (WAIS)III (4). assessspatialrecognitionmemory.Briefly,micewerehabituated
TheRBANSconsistsof12subteststhatareusedtocalculate for4dtoanemptymousecagefor10mineachday.Onday5,
five index scores and a total score. Test indices are Immediate twoidenticalobjectswereplacedonoppositeendsoftheempty
Memory (comprising List Learning and Story Memory tasks), cage,andthemousewasallowedtofreelyexploretheobjectsfor
Visuospatial/Constructional (comprising Figure Copy and Line 10min.Aftera1-hdelay,oneobjectwasmovedtoadifferent
locationinthecage,andthemousewasallowedtoexplorefor
Orientation tasks), Language (comprising Picture Naming and
5min.Thepercenttimeneartheobjectatthenovellocationwas
Semantic Fluency tasks), Attention (comprising Digit Span and
calculated as the time near the novel location divided by total
Coding tasks), and Delayed Memory (comprising List Recall,
time near novel and old location.
Story Recall, Figure Recall, and List Recognition tasks). Each
Sensorimotorgating.SensorimotorgatingwasassessedusingPPIof
index score is expressed as an age-adjusted standard score with
a mean of ∼100 and an SD of ∼15. The index scores were the acoustic startle (San Diego Instruments Inc.). Mice were
acclimatizedtoa70-dBbackgroundnoisefor5min.Theywere
combined to yield an RBANS Total score, which is a measure
then given 10 presentations each of a 120-dB pulse and 0-dB
of overall cognitive functioning. Trails A requires an individual
pulse. This was followed by 5–6 presentations in randomized
to draw lines sequentially connecting 25 encircled numbers dis-
order of a 120-dB pulse, 0-dB pulse, or the following prepulses
tributed on a sheet of paper; the score is based on the time to
followed by the 120-dB pulse: 74, 78, 82, 86, and 90 dB. The
completethetask.TrailsAisatestofvisualscanningandmotor
intervalsbetweeneachpresentationvariedfrom10to19s.PPI%
speed.TheInformationsubtestoftheWAISisatestofgeneral wascalculatedby[100–(meanstartleamplitudeofeachprepulse/
knowledge, including questions about geography and literature. meanstartleamplitudeof120dBpulse)]×100.MeanPPI%was
Forthelattertwotests,scoresareexpressedasanage-adjusted
calculated by averaging all PPI% values for presentations of all
scaledscorewithameanof10andanSDof3.Forthepurposesof
prepulsesforeachexperimentalgroup.
calculatingoddsratios,alowperformanceontheRBANStestswas
Passiveavoidance.Associativelearningandmemorywereevaluated
defined as less than or equal to 80 and low performance on the
usinga2-dpassiveavoidancetest(SanDiegoInstrumentsInc.).
othertestsasperformancebelowthe25thpercentile(4,5).
Onday1,micewereplacedinalitcompartmentwiththegateto
thedarkcompartmentclosed.Aftera30-sdelay,thegateopened,
MonitortheBehaviorofMice.Behavioraltestswereperformedon
allowingthemousetocrosstothedarkcompartment.Oncethe
miceinoculatedwithChlorellaheliozoae/ATCV-1(exposed)and
mousecrossedover,thegateautomaticallyclosed,andaftera3-s
C.heliozoae(control)between6 and 22 wkpostinoculation.The
delay, a 0.3-mA shock was administered for 3 s. Twenty-four
tests were performed in the following order: novelty-induced ac-
hourslater, the mouse was again placed inthe litcompartment
tivityinopenfield,Y-maze,objectplacement,dark–lightbox,new
withthe gateshut. Aftera5-sdelay, thegate opened.Thetrial
object recognition or location, prepulse inhibition (PPI) of the
endedeitherwhenthemousecrossedtothedarkcompartmentor
acousticstartle,andpassiveavoidance.
once10minelapsed.Oneachday,thelatencyinsecondsforthe
Novelty-inducedactivity.Novelty-induced activity inthe open field
mouse to cross from the light to dark compartment was auto-
was assessed over a 30-min period using activity chambers with
matically recordedandused inthe analysis.
infrared beams (San Diego Instruments Inc.), as previously de-
scribed(6). StatisticalAnalysesofBehavioralStudies.Thebehavioraldatawere
Spatial recognition in the Y-maze. Spatial recognition memory was analyzedwithone-wayanalysisofvariance(ANOVA)foralltests
evaluated in a Y-maze as described by Melnikova et al. (7). In exceptPPI.ThePPIdatawereanalyzedusingtwo-wayrepeated
brief,onearmofthemazewasblockedandamousewasallowed measures ANOVA with treatment as a between-subject factor
to freely explore the two open arms for 5 min. After a 20-min andPPIasawithin-subjectsfactor.Wedidnotusesexofanimals
delay, the block was removed and the mouse was allowed to as an independent variable, as our analyses detected no sex-
freelyexploreallthreeopenarmsfor5min.Thepercentageof dependenteffectsinthetestsdescribed.Ifthedatadidnotpass
timeandvisitsintothenovel(previouslyblocked)armduringthe tests for normality or equal variance, the data were rank-trans-
first2 minof the5-min trial was analyzed. formedbefore further statistical analysis.
Dark–light box. Anxiety was evaluated using a dark–light box
(Colbourn Instruments). Mice were placed in the transparent Metagenomic Sequencing. DNA samples from 33 individuals (two
sideof the boxandallowed to freelymove between the dark and independentexperimentswith17and16individuals)wereanalyzed
lightchambersfor5min.Thelatenciestocrossbetweenchambers bymetagenomicsequencing.Thedemographicinformationon
wereautomaticallyrecordedusingGraphicStatev3.03. these individuals is reported in Table S1A. The method used
Yolkenetal.www.pnas.org/cgi/content/short/1418895111 1of9
forthesequencingispresentedasfollows.Atotalof75–100ng differentially expressedgenesunder variousconditions,asingle
of DNA was used for paired-end library generation using the expression value was assigned for each transcript, including all its
NugenUltralowDRMultiplexSystem(NuGEN)followingthe exons. One-wayANOVAwasused todetect statisticallysignificant
manufacturer’s instructions. The libraries were purified and changesingeneexpressionbetweensamplesfrommiceinoculated
analyzed on the Bioanalyzer (Agilent Technologies) to con- with C. heliozoae/ATCV-1 and control mice inoculated with C.
firm size and concentration. The purified libraries were se- heliozoaealone.Transcriptswithadifferenceofatleast2SDsin
quencedusingIlluminaHiSeq,whichgenerated∼200,000,000 either direction between the ATCV-1–exposed and control mice
paired-end reads of 100 nucleotides. were selected for pathway analyses (12).
Sequencereadswerefilteredtoremovelow-qualitysequences,
resulting in a minimum length of 60 nucleotides. To evaluate PathwayAnalysis.Network, function, andpathway analyses were
putative viruses associated with the human throat sequence generatedusingIngenuityPathwaysAnalysis(IngenuitySystems),
reads,human,bacteria,fungi,andparasitesequencereadswere whichfacilitatestheinterpretationofmicroarraydatabygrouping
removedbybioinformaticfilteringasfollows:sequencereadswith differentially expressed genes into known functional pathways.
homology to human samples were removed in two stages. The These analyses identified statistically increased representations
firststageusedtheprogramBowtie(bowtie-bio.sourceforge.net/ ofthedifferentiallyexpressedgenesinbiologicallyrelevantpro-
index.shtml). A sliding window approach was used to align a cesses(13).Genesthatshoweddifferentialexpressionofgreater
40-base-pairsubsequence fromthereadstothehumangenome than2SDsbetweeninoculatedandcontrolmicewerecompared
Build 37 (www.snpedia.com/index.php/GRCh37). During each with those genes that did not, using the Fisher’s exact test to
iterationofthisprocedure,readsmappingtothehumangenome identify the differentially expressed genes’ pathways for review
were removed from the analysis and subsequences used for ofpotentialbiologicalfunction.BasedonitscuratedKnowledge
alignment were offset by five bases. The second filtering of hu- Base (MAP Molecule ActivityPredictor; www.ingenuity.com/
man sequences used CLC Genomics Workbench Version 6 products/ipa/ipa-summer-release-2014), Ingenuity Pathways
(www.clcbio.com) using a reference set of sequences based on Analysisfurtherpredictedwhetherthegenes’observedlevelsof
the human genome Build 37 with the following settings: length altered transcription were in accordance with regulatory rela-
fraction, 0.4; similarity, 0.4. Sequence reads that were not re- tionships from the literature; for example, elevated expression
moved by this subtraction were filtered sequentially to remove of gene A (a known inhibitor of gene B) is observed together
bacterial, fungal, and protozoan sequences by matching to ap- with reduced expression of gene B. Due to the exploratory na-
propriate National Coalition Building Institute Reference Se- ture of this study, pathways with P values ≤ 0.05 were selected
quence(RefSeq)databases(www.ncbi.nlm.nih.gov/refseq/).The for inclusion.
remaining sequences, which consisted of <1% of the starting se-
quences, were then mapped to the RefSeq complete set of viral MeasurementofAntibodiestoATCV-1andRelatedChlorovirusesin
genomes (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral/) using CLC MouseBloodSamples.
GenomicsWorkbenchVersion6withthefollowingsettings:length Enzymeimmunoassays.IgG antibodies to ATCV-1were measured
fraction, 0.8; similarity, 0.8. Sequences homologous to ATCV-1 by ELISA using variations of previously described procedures.
(reference sequence NC_008724.1) by this analysis were further Highly purified virion stocks containing ∼1011 PFU/mL were
mappedtotheATCV-1genomeusingCGView(11). diluted1:1,000in50μLcarbonatebufferandcoatedovernightat
4°Con96-wellpolystyreneflatbottomMaxiSorpplates(Nunc;
RNA Extraction. Following completion of the behavioral experi- Thermo Fisher Scientific). Plates were blocked for 1 h at 37 °C
ments,themicewerekilledandbrainswereremovedandplaced with Starting Block (Thermo Scientific). Plates were then in-
on ice. The hippocampus was dissected, placed into RNAlater cubated with a 1:1,000 dilution of the mouse test serum in du-
RNAstabilizationreagent(Qiagen),andstoredat−80°C.Total plicatewells,incubatedfor1hat37°C,washed,andincubated
RNA was isolated from either the left or right hippocampus with peroxidase-conjugated goat–anti-mouse IgG for 45 min at
usingmiRNeasyQiagenminikit(cat.no.217004)followingthe 37 °C (Southern Biotech). A 2,2’-azino-di-(3-ethylbenzthiazo-
manufacturer’s protocol. To remove genomic DNA carryover, line-6-sulfonate) hydrogen peroxide solution (KPL Protein Re-
RNA samples were treated with DNase for 20 min at 37 °C search Products) was added for color development, and
using a TurboDNA-free kit from Ambion (cat. no. AM1907). absorbance was measured at 405 nm, with a reference wave-
Samples were assessed for RNA quality and concentration by length of 490 nm, in an automated microtiter plate reader
TapeStation 2200(Agilent Technologies). Based on these mea- (Molecular Devices), with the results expressed as absorbance
surements,24sampleswereselectedforanalysis.Theseincluded units.AsamplewasconsideredpositiveforantibodiestoATCV-1
samples from 16 mice gavaged with C. heliozoae/ATCV-1 and ifitgeneratedasignalinthewellscoatedwithATCV-1thatgave
from eight mice gavaged with C. heliozoae alone. anabsorbancevalueofatleast0.4units.
MeasurementofantibodiesbyWesternblotting.C. heliozoae infected
Microarray Analysis. RNA transcript levels were quantified by with ATCV-1 and uninfected C. heliozoae were added to SDS
microarray analyses. RNAs were amplified into cDNA and Laemmlibufferandheatedto95°C,diluted1:10,andloadedon
biotinylated by in vitro transcription with Affymetrix reagents, a precast NuPAGE Novex 4–12% Bis·Tris gel (Life Technolo-
using the Whole Transcript Sense Target Labeling protocol as gies). Proteins were resolved through SureLock gel electropho-
describedintheAffymetrixmanual(www.affymetrix.com/support/ resis,andthegelswerestainedwithSimplyBlueSafeStain(Life
technical/product_updates/wt_1_1_assay.aff). Biotinylated cDNAs Technologies) to visualize protein amounts. Proteins were
were purified, fragmented, and subsequently hybridized to Affy- transferred to nitrocellulose using iBlot dry transfer technol-
metrixGeneChipMouseGene2.0STarrays. ogy (Life Technologies). Membranes were incubated over-
night at 4 °C with Starting Block (Thermo Scientific).
Data Normalization and Statistical Analysis for Microarray Tran- Following 1 h incubation with a 1:1,000 dilution of the test
scriptomics.AffymetrixCELfiles,containingtherawGeneChipdata, sampleofmouseserum,theblotswerewashedandincubatedfor
were prepared using GeneCp Command Console software. These 45 min with goat–anti-mouse IgG conjugated to alkaline phos-
datawereextractedandnormalizedwithGenomicsSuitev6.6(Partek phatase (Southern Biotech). Following another incubation, the
Inc.) software using the Robust Multichip Analysis algorithm. One blots were washed and developed with an alkaline phosphatase-
C.heliozoaeinoculatedcontrol(IDno.650)hadRNAthatwas based conjugation kit (BioRad Life Science). For reference, we
largelydegradedandwasomittedfromthefinalanalyses.Todetect also used a 1:500 dilution of a rabbit polyclonal antibody gener-
Yolkenetal.www.pnas.org/cgi/content/short/1418895111 2of9