DTIC ADA520746: Rapid Identification of Vector-Borne Flaviviruses by Mass Spectrometry

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MolecularandCellularProbesxxx(2010)1e10
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Molecular and Cellular Probes
journal homepage: www.elsevier.com/locate/ymcpr
Rapid identification of vector-borne flaviviruses by mass spectrometry
Rebecca J. Grant-Kleina, Carson D. Baldwina, Michael J. Turella, Cynthia A. Rossia, Feng Lib,
Robert Lovarib, Chris D. Crowderb, Heather E. Matthewsb, Megan A. Roundsb, Mark W. Eshoob,
Lawrence B. Blynb, David J. Eckerb, Rangarajan Sampathb, Chris A. Whitehousea,*
aUnitedStatesArmyMedicalResearchInstituteofInfectiousDiseases,1425PorterStreet,FortDetrick,MD21702,USA
bIbisBiosciences,1891RutherfordRd.,Carlsbad,CA92008,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: FlavivirusesareahighlydiversegroupofRNAvirusesclassifiedwithinthegenusFlavivirus,familyFla-
Received5February2010 viviridae.Mostflavivirusesarearthropod-borne,requiringamosquitoortickvector.Severalflaviviruses
Accepted5April2010 arehighlypathogenictohumans;however,theirhighgeneticdiversityandimmunologicalrelatedness
Availableonlinexxx
makesthemextremelychallengingtodiagnose.Inthisstudy,wedevelopedandevaluatedabroad-range
Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/elec-
Keywords: trospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was
Ibis-T5000 evaluatedwithapanelof13differentflaviviruses.Allsampleswerecorrectlyidentifiedtothespecies
Electrosprayionization
level.Todeterminethelimitofdetectionforthemosquito-borneprimersets,serialdilutionsofRNAfrom
Tick-borne
Mosquito-borne West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2
Arbovirus plaque-formingunits/mL.Analysisofflavivirusesintheirnaturalbiologicalbackgroundincludedtesting
Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately
identified the virus within infected mosquitoes, and we determined the average viral genome per
mosquitotobe2.0(cid:1)106.Usinghumanblood,serum,andurinespikedwithWNVandmousebloodand
braintissuesfromKarshivirus-infectedmice,weshowedthattheseclinicalmatricesdidnotinhibitthe
detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks
collectedfromsitesinNewYorkandConnecticut.Wefound16/322(5%infectionrate)tickspositivefor
deertickvirus,asubtypeofPowassanvirus.Insummary,wedevelopedasinglehigh-throughputFla-
vivirusassaythatcoulddetectmultipletick-andmosquito-borneflavivirusesandthusprovidesanew
analyticaltoolfortheirmedicaldiagnosisandepidemiologicalsurveillance.
PublishedbyElsevierLtd.
1. Introduction dengue viruses serotypes 1e4 (DENV 1e4), yellow fever virus
(YFV),WestNilevirus(WNV),Japaneseencephalitisvirus(JEV),and
Flaviviruses are single-stranded, positive-sense, RNA viruses St.Louisencephalitis(SLEV).
classified in the genus Flavivirus within the family Flaviviridae. Virusesinthetick-borneencephalitisvirus(TBEV)complexare
Therearemorethan50virusspecieswithinthegenus,andmost significant humanpathogens in various parts of Europe and Asia
arearthropod-borne,beingtransmittedtovertebratesbyinfected [15] and have been defined geographically and phylogenetically
mosquitoes or ticks [16]. Phylogenetic analysis has demonstrated intotheEuropean,FarEastern,andSiberiansubtypes[9].Several
threemajorgroupsofflaviviruses,comprisingthemosquito-borne, tick-borne flaviviruses can also cause hemorrhagic disease.
tick-borne,andno-known-vectorclades[21].Oftheknownflavi- Important examples of these viruses include Omsk hemorrhagic
viruses, approximately 50% are recognized human pathogens fevervirus(OHFV)inRussia,KyasanurForestdiseasevirus(KFDV)
causing fever, encephalitis, or hemorrhagic disease; however, for inIndia,andthecloselyrelatedAlkhurmahemorrhagicfevervirus
manyoftheothers,theirpathogenicpotentialhasnotbeenwell- (AHFV),whichhasbeenararecauseofhemorrhagicfeverinSaudi
studied [16]. Important mosquito-borne flaviviruses include Arabia since its initial description in 1995 [36]. Powassan virus
(POWV)istheonlyrecognizedtick-borneflaviviruspathogenicto
humans in the Americas [6]. It occurs in parts of eastern Russia,
* Correspondingauthor.Tel.:þ13016192098;fax:þ13016192492. Canada,andinisolatedfociinthenortheasternandnorth-central
E-mailaddress:[email protected](C.A.Whitehouse). United States. Cases of encephalitic disease caused by this virus
0890-8508/$eseefrontmatterPublishedbyElsevierLtd.
doi:10.1016/j.mcp.2010.04.003
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
CellularProbes(2010),doi:10.1016/j.mcp.2010.04.003
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Rapid identification of vector-borne flaviviruses by mass spectrometry
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2 R.J.Grant-Kleinetal./MolecularandCellularProbesxxx(2010)1e10
appear to be on the increase in the United States [17]. Deer tick 2. Materialsandmethods
virus (DTV), which is closely related to POWV, was first isolated
from Ixodes scapularis ticks in 1997 from North America [14,32]. 2.1. Viralisolates,plaqueassay,andRNAextraction
DTVisnowconsideredageneticsubtypeofPOWVandwasrecently
showntobeacauseoffatalencephalitis[31]. ThevirusesusedinthisstudywerepartoftheU.S.ArmyMedical
Development of broad-range flavivirus diagnostic assays has ResearchInstituteofInfectiousDiseases(USAMRIID)virusculture
been problematic largely because of the high degree of genetic collection,andtheircharacteristicsarelistedinTable1.RNAfrom
diversityandimmunologicalcross-reactivityamongtheseviruses. virus cultures and from pooled mosquitoes was extracted using
Many molecular amplification assays for flaviviruses have been TRIzol-LS(cid:1) (Invitrogen Corp., Carlsbad, CA) according to the
developedovertheyears[22].Severalattemptstodevelopbroad- manufacturer’s instructions. RNAwas quantified and checked for
rangeoruniversalflavivirusdetectionassayshavealsobeenmade, qualitybymeasuringabsorbanceat260and280nm.Sampleswere
typically using RT-PCR with degenerate primers targeted to storedat(cid:3)70(cid:4)Cuntilused.
conserved regions of the genome. However, these assays require
either the sequencing of the resulting amplicons [2,24,26] or 2.2. Tickcollections
analysis by restriction digestion of the amplicons [13]. While the
use of mass spectrometry as a diagnostic tool has made great AdulttickswerecollectedbyflaggingfrommultiplesitesinNew
strides in recent years [11], it has mostly been used for bacterial YorkandConnecticut.Tickswerehomogenizedusingacombina-
identificationbytheexaminationofproteinorlipidprofilesusing tion of large and small yttria-stabilized zirconium oxide beads
matrix-assistedlaserdesorptionionization-time-of-flight(MALDI- (GlenMills,Clifton,NJ)andtotalnucleicacidswereextractedusing
TOF).Incontrast,theIbisT5000(IbisBiosciences,Inc.,asubsidiary amodificationoftheQiagenVirusMinElutekit(Qiagen,Valencia,
of Abbott Molecular) analyses DNA and determines the base CA)asdescribedelsewhere[5].
composition (AxGxTxCx) of PCR amplicons by using electrospray
ionization mass spectrometry (ESI-MS) (Fig. 1) [8,28]. In the 2.3. Mosquitoinoculationandvirusplaqueassay
present study, we developed an 8-primer-pair broad-range flavi-
virusassayusingRT-PCRcoupledwithESI-MS.Usingthisassay,we Aedes aegypti mosquitoes (Rockefeller strain) were obtained
tested multiple strains of viruses, representing both mosquito- from the Uniformed Services University of Health Sciences and
borne and tick-borne flaviviruses. In addition, to show that the inoculatedintrathoracicallywith0.3mLofavirussuspensioncon-
assayiscapableofdetectingvirusesinbiologically-andclinically- tainingabout105plaque-formingunits(PFU)/mL(101.5PFUinoc-
relevant matrices, we examined laboratory-infected mosquitoes, ulated per mosquito) of DENV1-4, or YFV. After inoculation,
bloodandbraintissuesfromlaboratory-infectedmice,andvirus- mosquitoes were held in cardboard containers in an incubator
spiked human clinical specimens. Furthermore, field-collected maintainedat26(cid:4)Cfor7daysandwereprovidedappleslicesdaily
ticks from several sites in the U.S. Northeast were tested for the asacarbohydratesource.Themosquitoesweretrituratedin0.6mL
presence of naturally occurring Flavivirus infection. Due to the of diluent containing 10% heat-inactivated fetal bovine serum in
increasedgeographicdistributionandseverityofdiseasecausedby Medium 199 with Earle’s salts, 5 mg of amphotericin B, 50 mg of
membersoftheFlavivirusgenus,novelmethodsfortheirdetection gentamicin,100unitsofpenicillin,and100mgofstreptomycinper
are critical for both vector surveillance efforts and clinical mL.Analiquotof0.1mLofeachsuspensionwasaddedto0.9mLof
diagnosis. diluentandfrozenat(cid:3)70(cid:4)Cuntiltestedforvirusbyplaqueassay.
Fig.1. TheoreticalspeciesresolutionforasubsetofmedicallyimportantFlavivirussequencesinGenBank.Mostmajorspeciesareclearlydifferentiatedfromeachother.Additional
primerpairsdesignedtospecificspeciesgroupswillprovideaddedresolvingpowerwithinacluster(datanotshown).
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
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Table1
Flavivirusesusedinthisstudy.
Virusname Abbreviation Strain Origin/location/yearofisolation GenBankaccessionno.
Mosquito-borneviruses
Denguevirus1 DENV-1 Hawaii Human/Hawaii/1944 EU848545
Denguevirus2 DENV-2 S16803 Thailand/? NR
Denguevirus3 DENV-3 H87 Human/1956 M93130
Denguevirus4 DENV-4 H241 Human/1956 AY947539
Japaneseencephalitisvirus JEV Nakayama Human/Japan/1935 EF571853
St.Louisencephalitisvirus SLEV MSI-7 Sparrow/Mississippi/1975 EU076711
St.Louisencephalitisvirus SLEV TBH-28 Human/Florida/1962 EU090145
St.Louisencephalitisvirus SLEV Parton Human/Missouri/1933 EU084877
Tembusuvirus TMUV 157 Mosquito/1955 NR
WestNilevirus WNV NY99 Crow/NewYork/1999 NC_009942
Yellowfevervirus YFV
Tick-borneviruses
CentralEuropeansubtypeTBEVa TBEV-CE Hypr Human/w1944 EU303231
CentralEuropeansubtypeTBEV TBEV-CE Vergina Goat/1969 NR
FarEasternsubtypeTBEV TBEV-FE Sofjin Human/1937 AF013399
FarEasternsubtypeTBEV TBEV-FE Mass93 Human/1993 NR
Karshivirus KV UZ-2247 Ticks/Uzbekistan/? NR
KyasanurForestdiseasevirus KFDV W371 Human/India/1957 AF013385
Langat LGTV TP-21 Tick/Malaysia/1956 EU790644
Omskhemorrhagicfevervirus OHFV Belangul Human/1947 NR
Powassan POWV LB Human/Canada/1958 L06436
a Tick-borneencephalitisvirusgroup;NR,notrecorded.
Beforebeingtransportedfromthebiosafetylevel-3suite,1.5mLof all mosquito- and tick-borne flaviviruses, with four primer pairs
(cid:1)
TRIzol-LS was added to the remaining 0.5 mL of mosquito (VIR2215,VIR2217,VIR2211,andVIR2216)beingpan-Flavivirus,one
suspension,andthissamplewasthensplitinhalftoproducetwo primer pair (VIR2208) targeting all mosquito-borne flaviviruses,
1-mlaliquots.RNAwasextractedfromoneofthesesuspensionsas oneprimerpair(VIR2234)targetingallfourserotypesofDENV,and
describedabove.Thedilutedmosquitosuspensionswerethawed, two primer pairs (VIR1026 and VIR1028) targeting all strains of
serial10-folddilutionsmade,andthentestedbyplaqueassayon WNV (Table 2). The VIR2217 primer pair targets the RNA-depen-
LLC-MK-2 cell monolayerstodetermine the amountofinfectious dentRNApolymerase(RdRp,NS5)genethatisconservedacrossall
virus present. Methods were essentially identical to those knownflaviviruses.Foreachprimerregion,adatabaseofexpected
described by Gargan et al. [12], except that the second overlay, basecompositions[AGCT]fromallknownFlavivirussequencesin
containingneutralred,wasadded5,ratherthan4daysafterthe GenBankwasgenerated(datanotshown)andusedintheidenti-
initialoverlay.Plaqueswerecountedthefollowingday,andtiters fication and classification. All primers used in this study had
were expressed a log10 PFU per mL of mosquito suspension. For athyminenucleotideatthe50 endinordertominimizetheaddi-
quantitativeanalysis,infectedmosquitopoolscontainedasingle- tion of non-template adenosines during amplification using Taq
virus-infected mosquito and nine uninfected mosquitoes. For the polymerase[4].
mixed infected mosquito pools, two infected mosquitoes (each
containingadifferentvirus)werecombinedwitheightuninfected 2.7. One-stepRT-PCR
mosquitoes.
APerkinElmerJanusrobot(Waltham,MA)wasusedtoset-up
2.4. SpikingofhumanclinicalspecimenwithWNV each one-step RT-PCR reaction. All RT-PCR reactions were per-
formed in 40 ml reaction using 96-well microtiter plates and an
Purified WNV was diluted to a final concentration of Eppendorf(cid:1) Mastercycler(cid:1) thermocycler (Eppendorf, Hamburg,
1(cid:1)105PFU/mLinhumanblood,serum,orurine(Bioreclamation, Germany).EachRT-PCRreactionbufferconsistedof3.0UofAmpli
Inc., Liverpool, NY), or phosphate-buffered saline (PBS) control. Taq Gold (Applied Biossystems, Foster City, CA), 20 mM Tris (pH
Duplicatealiquotsof250mlofeachspikedsamplewereaddedto 8.3), 75 mM KCl, 1.5 mM MgCl2, 0.4 M betaine, 200 mM, dATP,
750mlofTRIzol-LS(cid:1),andtheRNAwasextractedasdescribedabove. 200 mM dCTP, 200 mM dTTP (each dNTP from Bioline USA, Ran-
dolph, MA), 200 mM 13C-enriched dGTP (Spectra Stable Isotopes,
2.5. TissuesfromKarshivirus-infectedmice Columbia, MD),10 mM dithiothreitol,100 ngof sonicated poly-A
DNA(SigmaCorp,StLouis,MO),and250nMofeachprimer.The
Karshivirus-infectedtissueswereobtainedfromsucklingmice followingPCRconditionswereusedtoamplifysequences:60(cid:4)Cfor
thatwereinoculatedsubcutaneouslywhen2-day-oldwithasuck- 5min,4(cid:4)Cfor10min,55(cid:4)Cfor45minfollowedby8cyclesof95(cid:4)C
lingmousepassageofthisvirusaspartofapreviousstudyonthe for30s,and48(cid:4)Cfor30s,and72(cid:4)Cfor30sfollowedby37cycles
tissuedistributionofthisvirus[34].ThepresenceofKarshivirusin of95(cid:4)Cfor15s,56(cid:4)Cfor20s,and72(cid:4)Cfor20s.TheRT-PCRcycle
the samples was previously confirmed by a Karshi virus-specific endedwithafinalextensionof72(cid:4)Cfor2minfollowedbya4(cid:4)C
quantitative real-time PCR assay performed on the Roche Light- hold.
Cyclerasdescribed[34].
2.8. InternalpositivecontrolRNA
2.6. Primerdesign
To determine the efficiency of the RT-PCR, each reaction con-
Eightprimerpairsweredesignedtotargetthevariousmembers tainedasyntheticinternalpositiveDNAcontrol(IPC).TheIPCwas
ofthegenusFlavivirus(Table2).Theassaywasdesignedtoamplify producedbyinvitrotranscriptionfromaT7promoterpresenton
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
CellularProbes(2010),doi:10.1016/j.mcp.2010.04.003
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Table2
RT-PCRprimersusedinthepan-FlavivirusESI-MSassay.
Primerpairname Genetarget Coverage Primercoordinatesa Orientation Primersequence
VIR2215 NS5 pan-Flavivirus 8874e8966 F 50-TAGCCGAGCCATCTGGTACATGTGG-30
R 50-TCTCTGGAAAGCCAGTGGTCTTCATT-30
VIR2217 NS5 pan-Flavivirus 8812e8907 F 50-TGTGTCTACAACATGATGGGAAAGAGAGA-30
R 50-TGCTCCCAGCCACATGTACCA-30
VIR2208 NS5 pan-mosquito-borneviruses 8971e9080 F 50-TCATTGAGTGGAGTGGAAGGAGAAGG-30
R 50-TCCCAGCCGGCTGTGTCATC-30
VIR2234 NS3 pan-dengueviruses 5270e5365 F 50-TCATGGATGAAGCACATTTCACAGATCC-30
R 50-TGAAGATCGCAGCTGCCTCTCCCAT-30
VIR2211 NS5 pan-Flavivirus 8885e8964 F 50-TCTGGTTCATGTGGCTGGGAGC-30
R 50-TCTGCCCAGCCAGTGGTCTTCATT-30
VIR2216 NS5 pan-Flavivirus 8865e8951 F 50-TGCCAAGGGAAGCAGGGCCAT-30
R 50-TGGTCTTCATTGAGGAATCCCAGAGC-30
VIR1026 NS5 WNV 10,135e10,237 F 50-TGGATAGAGGAGAATGAATGGATGGAAGAC-30
R 50-TCAGGCTGCCACACCAGATGTC-30
VIR1028 NS3 WNV 5696e5783 F 50-TCAAGATGGGGAATGAGATTGCCCTT-30
R 50-TACTCCGTCTCGTACGACTTTCTGTT-30
a CoordinatenumbersforWestNilevirus(WNV)primers(VIR1026andVIR1028)arebasedonWNVstrain956,GenBankaccessionnumberNC_001563.Allotherprimer
coordinatenumbersarebasedondenguevirustype2,GenBankaccessionnumberNC_001474.
aclonedsyntheticDNAtemplatecontainingthetargetregionsfor correctorganismclassificationandreducesthepossibilityoffalse
the eight primer pairs. The IPC was present in each reaction at positives[7].ForeveryRT-PCRreactionwell,thesignalamplitude
apre-determinedconcentration(100copies/RT-PCRreaction)and oftheIPCandthesamplewerecomparedandinterpretedtogive
actedasacalibranttodeterminetheRT-PCRassay’sefficiencyand quantitativeresults.
providesemi-quantitativeinformation.
3. Results
2.9. Massspectrometryandbasecompositionanalysis
3.1. Detectionofflaviviruseswithbroad-rangePCRprimerpairs
After PCR, approximately 30 ml of each RT-PCR reaction was
bound to a weak anion exchange matrix where a series of wash
Flavivirusesareageneticallydiversitygroupofvirusesandthus
steps removed salts and excess reaction reagents as previously
poses a major challenge for broad-range molecular detection
described[19].Afterclean-up,thepurifiedRT-PCRproductswere
assays.Thegoaloftheworkdescribedherewastodevelopanassay
elutedfromthestationaryphaseusingavolatilebuffer.TheBruker
thatwouldallowforthebroaddetectionofallthediversemembers
Daltonics microToF (Billerica, MA) mass spectrometer (MS) was
ofthisgroupofviruses,withemphasisonthemedicallyimportant
used for analyzing the purified DNA [18]. Products from each
vector-borne viruses. To accomplish this goal, a large number of
reactionwellwereindividuallysprayedintotheMSusingaLEAP
primerpairsweredesignedandtested(datanotshown).Ofthese,
autosampler (LEAP Technologies, Carrboro, NC). Internal mass
eightprimerpairs,whichtargettheNS5andNS3viralgenes,were
standards and plasmid calibrants were utilized to reach a mass chosenforthefinalassayformat(Table2).Fig.2showsamultiple
accuracyofabout5e10ppmandprovidedaccuratemeasurements
sequence alignment of the pan-Flavivirus primer pair, VIR2217,
with high-resolution mass spectra for each sample by previously againsttheknownsequencesofseveralflavivirusesinGenBank.For
described protocols [18]. Proprietary signal-processing software
eachprimerregion,adatabaseofexpectedbasecompositionswas
was used to deconvolute raw data from mass per charge. This
generated (data not shown). Several of the isolates used in this
molecular mass was then assigned to the amplicon’s empirical
studydidnothavegenomicsequencesinGenBankandthusbase
molecular mass and correlating base composition, which was compositions for the target amplicons were determined
matchedwith those in the system’s database. Usinga numberof
experimentally.
statisticalconsiderations andmulti-primerresults,the organisms
were identified [25]. Essentially, results are triangulated across
primer pairs to determine organism assignments. The processing 3.2. Detectionofdiverseflaviviruses
software, GenX, is designed to run in an automated fashion on
multiple PC systems from an input queue. Parallel processing The8-primerpairpan-flavivirusESI-MSassaywasevaluatedfor
provides an efficient means of increasing throughput; processing itsabilitytodetectapanelofdiverseflavivirusescomprisingboth
times are 15e45 min/plate dependingon spectral complexity. As mosquito- and tick-borne viruses, which included viral isolates
complementaryDNAstrandsarepresent,thisrestrictionisusedto where there was limited sequence data information in GenBank.
limit the possible choices of base compositions consistent with Isolateswithknownsequencesshowed100%matchestoexpected
molecular weight. These base compositions are then used as base compositions (Fig. 3). Primer pairs VIR2215 and VIR2217
hypotheses, fromwhich a spectral representation is modeled. At showed the broadest coverage and amplified all isolates (Fig. 3).
thispoint,organismsconsistentwiththeprimerpairusedinPCR Basecompositionsweretakenfromatleasttwoormoreamplicons
amplification are identified from the amplicon database. A joint forthevariousprimerpairstogivespeciesresolutionandfordis-
least-square algorithm is used to correlate potential organism tinguishingisolatesatthesubtypelevel.Wealsodeterminedthat
identifications across multiple primers, using a triangulation additionalviruspassagesinculturedidnotalterthebasecompo-
method. This computation process improves the confidence of sitionresults(seemultiplelotsofthesamevirusinFig.3).
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
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Fig.2. MultiplesequencealignmentoftheFlavivirusprimerpairVIR2217targetingtheNS5geneandcontainingover230differentsequencesrepresentingallknownflaviviruses.
Tworepresentativesequencesfromeachmajorcladewithinthisgenusareshownhere.Dotsinthecolumnrepresentidentityofeachtargetvirussequencetotheprimersequence
(toprow).Primerpaircoordinatesshownherearebasedonthedenguevirus2(GenBankaccessionnumberNC001474).
Fig.3. Basecomposition[AGCT]dataoftheRT-PCRampliconsgeneratedbythepan-FlavivirusESI-MSassay.Identicalbasecompositionswithinacolumnarethesamecolor.
Uniquebasecompositionsareshownwithwhitebackgrounds.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthis
article.)
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Asevidenceoftheassay’sabilitytodetectmixturesofvirusesin tissues were obtained from suckling mice that were inoculated
asample,apresumablypurelaboratorypreparationofOHFVwas subcutaneouslywhen2-days-oldwith virusaspartofaprevious
testedandfoundtobecontaminatedwithYFV.Examiningthebase studyonthetissuedistributionofthisvirus.RT-PCR/ESI-MSanal-
compositionforfourdifferentgenetargetamplicons(VIR2216only ysis clearly detected Karshi virus in both blood and brain tissues
generated a base composition for YFV) and the mass spectra andconfirmedhighlevelsofthisneurotropicvirusinthebrainof
generatedfortheforwardandreverseDNAstrandsof theampli- infected mice (Table 4) as was shown previously using a Karshi
cons, it was clear that there were two distinct DNA signatures, virus-specificquantitativereal-timeRT-PCRassay[34].
whichwereidentifiedasOHFVandYFVbytheFlavivirusRT-PCR/
ESI-MSassay(Fig.4).
3.6. Detectionofflavivirusesfromlaboratory-infectedmosquitoes
3.3. Sensitivityofthebroad-rangePCRprimerpairs
TheabilityoftheRT-PCR/ESI-MSassaytodetectFlavivirusRNA
from infected mosquitoes was tested by using a blinded set of
To determine the sensitivity of the mosquito-borne primer
laboratory-infected mosquitoes and uninfected controls. From 16
pairs, WNV was serially diluted 2-fold from a known titer and
differentmosquitopools,thecorrectviruswasidentifiedin15of
tested in replicates of 10. All six mosquito-borne primer sets
the samples (94% correct), highlighting the assay’s ability to
(VIR2215,VIR2217,VIR2211,VIR2216,VIR1026,VIR1028)had100%
distinguish between closely related DENV serotypes (Table 5).
sensitivity (10/10 reactions were positive) at 25 PFU/mL and all,
However,inonesample(W123)onlyaDENV3-infectedmosquito
exceptVIR2217,were100%sensitivedownto1.6PFU/mL(Fig.5).
waspresentinthesample,andthesystemalsoidentifiedDENV2.
Overall,theFlavivirusRT-PCR/ESI-MSassaycouldaccuratelyiden-
And another sample (W131) was a mixed pool containing both
tifyWNVdownto0.2PFU/mLwithVIR2216having50%sensitivity,
DENV1andDENV2;however,thesystemonlydetectedDENV2.
VIR2211 having 20% sensitivity, and VIR2215, VIR1026, VIR1028
Theonesamplethatwascompletelyincorrect(T136)wasanega-
having10%sensitivity(Fig.5).
tivecontrolcontainingonlyuninfectedmosquitoesandthesystem
identifieditascontainingWNV.NoWNVwasusedtoinfectanyof
3.4. TestingofWNVspikedclinicalmatrices
the mosquitoes; however, we cannot rule out possible contami-
nationofthesamplewithWNVRNAorPCRamplicon.
Totesttheassay’sabilitytodetectandcorrectlyidentifyafla-
viviruswithinrelevanthumanclinicalmatrices,WNVwasspiked
intospecimensofhumanblood,urine,andserumandtestedwith 3.7. QuantificationofDENVviralloadinindividual
the PCR/ESI-MS assay. The six mosquito-borne Flavivirus primer infectedmosquitoes
setshad100%agreementfordetectingandidentifyingWNVfrom
all three of the clinical matrices, which included blood, urine, EachreactionwellintheRT-PCR/ESI-MSassaycontainedanIPC
serum,andaPBScontrol.Eachofthebasecompositionsforeach to allow for quantitative analysis. Thus, we wanted to test the
primersetwere100%identicalforthefourdifferentsampleback- quantitativeabilityoftheassaytodeterminetheviralload(based
grounds(Table3). on genome equivalents) of DENV 1 present in individual virus-
infectedmosquitoesandcomparethattoplaquetiterofthesame
3.5. IdentificationofKarshivirusfromlaboratory-infectedmice mosquitoes.TenmosquitopoolsconsistingofoneDENV1-infected
andnineuninfectedmosquitoesweretestedinduplicateusingthe
To further evaluate the assay’s performance with Flavivirus- RT-PCR/ESI-MSassayandvirusplaqueassay.Themeannumberof
infectedmammaliantissues,Karshivirus-infectedbloodandbrain genomeequivalentspermosquitowasdeterminedtobe2.0(cid:1)106,
Fig.4. MassspectraofRT-PCRampliconsderivedfromanOmskhemorrhagicfevervirus(OHFV)stockculturecontaminatedwithYFV.Labelsandsignalsarecoloredaccordingto
thetheoreticalspectraforeachorganism:blue¼OHFV,green¼YFV.ActualspectrageneratedaretracedinblackandcorrespondtothesenseandantisenseDNAstrands.
(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
CellularProbes(2010),doi:10.1016/j.mcp.2010.04.003
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110
100
90
0) 80
1
=
n
e ( 70 VIR2215
st Nil 60 VIR2217
e VIR2211
W
of 50 VIR2216
n VIR1026
ctio 40 VIR1028
e
et
D 30
%
20
10
0
25 12.5 6.25 3.125 1.6 0.8 0.4 0.2 0
PFU/mL
Fig.5. Sensitivityofthesixmosquito-borneFlavivirusRT-PCRprimerpairs.FromaknownWestNilevirus(WNV)titer,theRNAwasseriallydilutedten-fold.Tenreplicateswere
performedtodeterminethesensitivityofeachdilutionwithascoreof100%indicatingthatallreactionsclearlyidentifiedWNV.
and the mean plaque titer was approximately 100-fold less at 4. Discussion
1.3(cid:1)104PFUpermosquito(Fig.6).
Withover70differentviralspeciesclassifiedinthegenusFla-
vivirus,broad-rangedetectionoftheseviruseshasbeenextremely
3.8. Screeningoffield-collectedticksforPowassanvirus problematic. Detection and identification of flaviviruses is made
even more complicated by the fact that these viruses are RNA
The pan-Flavivirus primer pairs, VIR2215 and VIR2217, were viruses,whichevolverapidly,andtherefore,subtlechangesintheir
used to screen field-collected I. scapularis ticks collected from genomes can rapidly make a once highly sensitive and specific
multiple sites in NewYorkand Connecticut during the 2008 and molecular based assay obsolete. In the current study, we have
2009tickseasons(Table6).Wefound16ticksoutofatotalof322 developedabroad-range(i.e.,pan-Flavivirus)RT-PCR/ESI-MSassay
tested (5% infection rate) positive for POWV and many of these that could rapidly identify and discriminate multiple species of
positivetickswerefurthercharacterizedashavingdeertickvirus, vector-borne flaviviruses in a single high-throughput assay. This
arecognizedsubtypeofPOWV.Distinctsignalscanbeseenforboth assay,performedontheIbisT5000system,usesstandardone-step
the forward and reverse strands of amplicon DNA obtained from RT-PCR with broad-range primers targeting the breadth of the
specimen G110708MR-5 using the pan-Flavivirus primer sets genus Flavivirus, followed by base composition determination
VIR2215 and VIR2217 (Fig. 7). Of note, the assay was able to usingatime-of-flightmassspectrometer.Inthisstudy,weshowed
distinguish three different POWV signatures based on base that the RT-PCR/ESI-MS assay accurately detected and identified
compositiondata(Table6).Approximately700nucleotidesofviral awiderangeofflavivirusesincludingmultipletick-andmosquito-
RNA from specimens G110708MR-5 and G082908CP-11 were borneviruses(Fig.3).UsingWNV,thesensitivityoftheassaywas
sequenced using the primers of Telford et al. [32]. The resulting determinedtobeapproximately2PFU/mL;however,detectionwas
sequences showed 97% identity to DTV, but only 87% identity to still possible with some of the primer sets, albeit at lower sensi-
POWV(datanotshown)indicatingthattheFlavivirusfoundwasthe tivities, down to 0.2 PFU/mL (Fig. 5). This limit of detection is
DTVsubtypeofPOWV.Onlyasingletickwasfoundtohaveathird consistentwiththosereportedforvarioustraditionalandreal-time
unique RT-PCR/ESI-MS base composition signature (specimen RT-PCR assays for WNV and other flaviviruses [22,23] and other
#082908CP-11),butwewereunabletoobtainsequenceinforma- mosquito-borne arboviruses [35]. It is also consistent with the
tiontoconfirmthevirussubtypeduetolimitedsamplevolume. sensitivityofapreviouslydevelopedRT-PCR/ESI-MSassayforthe
Table3
WestNilevirus(WNV)wasspikedintodifferenthumanclinicalmatricesataknownconcentrationof1(cid:1)105PFUpermLandtestedwiththePCR/ESI-MSassay.Corresponding
basecompositiondataareshownforeachofthemosquito-bornevirusprimersusedintheassay.Calculatedvaluesforthemeanlogarithm10genomeequivalentspermLfor
eachspecimentypearealsogiven.
Samplematrix GEa Primerpairs[AGCT]
2217 2215 2216 2211 1028 1026
Blood 3.8 [15153333] [26232717] [22202817] [24172712] [18152530] [17163436]
Serum 3.1 [15153333] [26232717] [22202817] [24172712] [18152530] [17163436]
Urine 3.7 [15153333] [26232717] [22202817] [24172712] [18152530] [17163436]
PBS 3.5 [15153333] [26232717] [22202817] [24172712] [18152530] [17163436]
a Meanlogarithm10genomeequivalentspermL.
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
CellularProbes(2010),doi:10.1016/j.mcp.2010.04.003
ARTICLE IN PRESS
8 R.J.Grant-Kleinetal./MolecularandCellularProbesxxx(2010)1e10
Table4 108
ComparisonofaKarshivirus-specificquantitativereal-timeRT-PCRassayaandthe
pan-Flavivirus RT-PCR/ESI-MS assay for testing of blood and brain tissues from 107
experimentally-infectedmice.
106
MIDouse Ttyispseue Dinafeycstpioonst- Qreuaal-nttiimtaetive pRaTn-P-FClRav/EivSiIr-uMsS quito 105
RT-PCR(GEb) SystemID GEb mos 104
102 Blood 1 5.3 Pos-Karshivirus 5.8 er
111131449 BBBrrraaaiiinnn 596 11019...302 PPPooosss---KKKaaarrrssshhhiiivvviiirrruuusss NNNDDDc Titer p 110032
a Thequantitativereal-timeRT-PCRassayusedherewasdevelopedandpublished 101
previously[34].
b Meanlogarithm10genomeequivalentspermLofbloodorgoftissue. 100
c Notdeterminedduetothehighgenomeequivalentspresentinthebraintissue GE PFU
wereabovethelinearityforquantitativeanalysisoftheESI-MSassay.
Fig.6. Viralloadperindividualmosquitoasestimatedbythequantitativepan-Flavi-
virusESI-MSassayandplaqueassay.SevenAedesaegyptimosquitoeswereinoculated
alphaviruses (assuming that 30 genome equivalents is approxi- intrathoracicallyinoculationwithapproximately101.5PFUofDENV-1.Sevendaysafter
matelyequalto3PFU)[10]. inoculation,mosquitoeswereprocessedasdescribedintheMaterialsandmethods
One benefit of the pan-Flavivirus RT-PCR/ESI-MS assay is its andweretested.Horizontallineindicatesmeantiter.GE,genomeequivalents;PFU.
abilitytoidentifymorethanonetypeofvirusinasample.Thiswas
evidentwhilewewereusingtheassaytoperformqualitycontrolof
Thoughitwasimportanttovalidatetheassaywithwell-char-
ourpurifiedvirusstocksforourculturecollection.Forexample,one
acterizedsamplesthathavebeentestedonotherdiagnosticplat-
sampleof OHFVwasclearlycontaminated with YFV(Fig. 4). This
forms,thedetectionofvirusessuchasTembusuandLangatviruses
characteristicoftheassaywouldalsohaveclearbenefitsforusein
demonstratestheassay’sabilitytoidentifylessknownflaviviruses
vector surveillance where two or more flaviviruses are co-circu-
wherelittleornosequencedataareavailable.Thisattributeofthe
latingwithinthesamegeographicregion,asisthecaseforWNV
assaywillbeparticularlyusefulindetectingrapidlyevolvingRNA
andDENVorformultipleserotypesofDENVforexample.Because
viruses, orthose thatarecompletelynovel, especially fromfield-
mosquitoes are often tested in pools of up to 50 or even 100, it
collectedspecimens.Thehigh-throughputandbroadnatureofthe
wouldbeverypossibletohavemorethanone-infectedmosquito
Ibis T5000 platform makes it highly amendable to public health
within any given pool. Likewise, individuals living in geographic
laboratory surveillance work. In particular, the previously devel-
regions were multiple arboviruses co-circulate, are at risk of co-
opedpan-AlphavirusESI-MSassaycouldbecombinedwiththepan-
infections,whichhasbeendocumentedpreviously[29,30].
Flavivirusassaydevelopedinthisstudyintoasingle“Arboviruskit”
Wefurtherdemonstratedthequantitativenatureoftheassayby
andusedformosquitoand/ortick-bornearbovirussurveillance.To
determiningoftheaveragenumberofviralgenomesofDENV1per
demonstratetheusefulnessoftheFlavivirusassayinvector-borne
individual laboratory-infected Ae. aegypti mosquito. The average
pathogensurveillance,wescreened322field-collectedI.scapularis
viral load per infected mosquito was calculated to be 2.0 (cid:1) 106
tickscollectedfromNewYorkandConnecticutforthepresenceof
genomesor1.3(cid:1)104PFU(Fig.6),whichisconsistentwithprevious
flaviviruses.Whileseveralmedicallyimportanttick-borneFlavivi-
studies [1,20]. Furthermore, our finding that the number of
rus are known to occur and cause severe neurological disease in
genomes per individual infected mosquito was about 100-fold
Europe and Asia (e.g. tick-borne encephalitis virus), POWV is the
higher than the plaque titer is consistent with other studies
only recognized tick-borne Flavivirus in the U.S. [6,14]. This is
showingthatPFUwasconsistentlylowerthanRNAcopynumberby
consistentwiththefactthatDTV,asubtypeofPOWV,wastheonly
2e3 log10 for both cell culture and mosquitoes infected with Flavivirus found among the ticks tested. Furthermore, we
DENV[27].
Table5
Assayperformancefordetectionofflavivirusesfromablindedpaneloflaboratory-infectedmosquitoes.Basecompositiondataareshownforeachoftheprimersetsinthe
FlavivirusRT-PCR/ESI-MSassay,alongwiththesystemidentification.Eachsamplewasassayedintriplicate.
CodedSampleID ActualSampleID PrimerpairsBasecomposition[AGCT]
2217 2215 2216 2211 1026 2234 2208 SystemID
W121 DENV1 [36321018] NDa ND [16251722] ND [26282418] [37331921] DENV1
W122 DENV2 [33341316] [22242324] [21261921] [17241821] [38331418] [30262119] [34401719] DENV2
W123 DENV3 [34321020] [22242324] [21261921] [15271919] ND [28272021] [32362022] DENV2,3
W124 DENV4 [30341317] [19291926] [20291820] [14281424] ND [26272122] [34381523] DENV4
W125 NCb ND ND ND ND ND ND ND Neg.
W126 DENV1 [36321018] [21262125] ND [16251722] ND [26282418] [37331921] DENV1
W127 DENV2 [33341316] [22242324] [21261921] [17241821] ND [30262119] [34401719] DENV2
W128 DENV3 [34321020] [20282322] ND [15271919] ND [28272021] [32362022] DENV3
W129 DENV4 [32341218] [19291926] ND [14281424] ND [26272122] [34381523] DENV4
W130 NC ND ND ND ND ND ND ND Neg.
W131 DENV1,2 [33341316] [22242324] [21261921] [17241821] [38331418] [30262119] [37331921] DENV2
T135 YFV [30331318] [19302123] [20301621] [14291621] ND ND ND YFV
T136 NC [28301818] ND [17282022] [12271724] [36341617] ND ND WNV
T137 DENV1 [36321018] [19302123] [20301621] [16251722] ND [26282418] [37331921] DENV1
T138 DENV1 [36321018] [21262125] [20301621] [16251722] ND [26282418] [37331921] DENV1
315e9 NC ND ND ND ND ND ND ND Neg.
a Nodetection.
b Negativecontrol.
Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
CellularProbes(2010),doi:10.1016/j.mcp.2010.04.003
ARTICLE IN PRESS
R.J.Grant-Kleinetal./MolecularandCellularProbesxxx(2010)1e10 9
Table6 fromNew England in 1995, theyfound onlya 0.4%infection rate
Characterizationoffield-collectedIxodesscapularisticks analyzedwiththepan- among 465 I. scapularis ticks collected from Connecticut and
FlavivirusESI-MSassay.
Massachusetts[32].MorerecentdatafromanorthernWisconsin
TickID Collection ESI-MS Genome Basecomposition[AGCT] focus shows a 1.3% infection rate among 1335 ticks tested [3]. In
location result copyno. VIR2215 VIR2217 a 2009 survey of I. scapularis collected from several locations
surroundingNewYorkCity,Tokarzetal.founda2.0%infectionrate
120208MR-Bridgeport,CT DTV 200 1925232627361419
3 forPowassanvirususingaMassTagPCRapproach[33].Although
120308MR-Bridgeport,CT DTV 300 1925232627361419 more studies are needed, our data suggest that the prevalence
9 Powassan virus, and particularly the DTV subtype, may be
082908CP- Connetquot,NY POWVa >330 1925222726371419
increasingintheU.S.northeast,andthisvirusmayemergeasan
11
082608CP- ShelterIsland, DTV >330 1925222727361419 importantpublichealthconcerninthefuture.
33 NY In conclusion, the ability of the pan-Flavivirus RT-PCR/ESI-MS
082708CP- ShelterIsland, DTV >330 1925222727361419 assaytorapidlyandsensitivelyidentifyknownandemergingfla-
5 NY vivirusesis critical fordiseasesurveillance andforadvancingthe
110608MR-Westchester DTV >330 1925222727361419
molecular diagnostic field past single-virus detection assays. We
6 county,NY
110608MR-Westchester DTV >330 1925222727361419 have shown that this assay has the ability to be a broad-range
8 county,NY detection tool for known and rare flaviviral species that cause
110608MR-Westchester POWVa 12 NDb 27361419 humandiseaseandcouldbenefitclinicaldiagnosticsorstudieson
9 county,NY
theecologyandepidemiologyofthisimportantgroupofviruses.
110708MR-Westchester POWVa 23 ND 27361419
2 county,NY
110708MR-Westchester POWVa 43 ND 27361419
4 county,NY Acknowledgements
110708MR-Westchester DTV >330 1925222727361419
5 county,NY TheauthorsthankDr.StuartNichol,SpecialPathogensBranch,
110708MR-Westchester POWVa 34 ND 27361419
Centers for Disease Control and Prevention, Atlanta, Georgia for
6 county,NY
110708MR-Westchester POWVa >330 ND 27361419 helpfuldiscussionsandforsupplyingtick-borneencephalitisvirus
14 county,NY isolatesforinitialtestingoftheassay.Theauthorsacknowledgethe
110708MR-Westchester POWVa 108 ND 27361419 DefenseAdvancedResearchProjectsAgency(DARPA),theDepart-
15 county,NY
mentofHomelandSecurity(DHS)contractnumberW81XWH-05-
052008CC- Bridgeport,CT DTV 79 1925222727361419
C-0116,theU.S.DefenseThreatReductionAgency(DTRA)(USAM-
22
061009MR-ShelterIsland, POWVa 13 ND 27361419 RIIDResearchPlan#114538)forfinancialsupport.Additionally,the
1 NY assay development and testing has been funded in part with
a Due to limited specimen DNA extract volume, the POWV subtype was not FederalfundsfromtheNationalInstituteofAllergyandInfectious
determined. Diseases,NationalInstitutesofHealth,DepartmentofHealthand
b Nodetection. Human Services, under Contract No. HHSN266200400100C. This
research was performed while R.J.G.-K. held a National Research
CouncilResearchAssociateshipAwardatUSAMRIID.FortheFlavi-
determineda5%infectionrateamongtheI.scapularistestedfrom
virussurveillanceoffield-collectedticks,weacknowledgetheLyme
the selected collection sites in New York and Connecticut. Previ-
DiseaseAssociation,theTamiFund,andtheNationalInstitutesof
ously published tick infection rates were much lower than our
Health,NationalInstituteofAllergyandInfectiousDiseasesgrant
findings. For instance, in the original publication describing DTV
#1R43AI077156-01forfinancialsupport.
Opinions, interpretations, conclusions, and recommendations
arethoseoftheauthorsandarenotnecessarilyendorsedbytheU.S.
Army,DepartmentofDefense,ortheNationalInstitutesofHealth.
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Pleasecitethisarticleinpressas:Grant-KleinRJ,etal.,Rapididentificationofvector-borneflavivirusesbymassspectrometry,Molecularand
CellularProbes(2010),doi:10.1016/j.mcp.2010.04.003