a novel pan-flavivirus detection and identification assay based...

Research ArticleA Novel Pan-Flavivirus Detection and Identification AssayBased on RT-qPCR and Microarray

Ariel Vina-Rodriguez1 Konrad Sachse2 Ute Ziegler1 Serafeim C Chaintoutis13

Markus Keller1 Martin H Groschup1 andMartin Eiden1

1 Institute for Novel and Emerging Infectious Diseases Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health)Insel Riems Greifswald Germany2Institute of Molecular Pathogenesis Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health) Jena Germany3Diagnostic Laboratory Department of Clinical Sciences School of Veterinary Medicine Faculty of Health SciencesAristotle University of Thessaloniki Thessaloniki Greece

Correspondence should be addressed to Martin Eiden martineidenflide

Received 20 December 2016 Accepted 7 February 2017 Published 24 May 2017

Academic Editor Satish B Nimse

Copyright copy 2017 Ariel Vina-Rodriguez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economicallyimportant animals While RT-PCR protocols for specific detection of most Flavivirus species are available there has been also ademand for a broad-range Flavivirus assay covering all members of the genus It is particularly challenging to balance specificityat genus level with equal sensitivity towards each target species In the present study a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed Validation experiments confirmed that the RT-qPCRexhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested These PCR products are subjectedto hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species Thisassay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples and it performedsuccessfully in international External Quality Assessment of NAT studies Twenty-six Flavivirus strains were tested with the assayshowing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs As an exampletest results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented

1 Introduction

The genus Flavivirus contains nearly 70 recognized virusesmany of which infect humans and economically importantanimals [1] Flaviviruses such as Dengue virus (DENV) [2]and Yellow fever virus (YFV) [3] have been a commoncause of devastating diseases in tropical and less developedcountries but in recent years the emergence of flaviviralzoonoses was observed worldwide Examples include theoccurrence of West Nile virus (WNV) in the United States[4] Japanese encephalitis virus (JEV) in Australia [5] andUsutu virus (USUV) [6]WNV [7] andDENV [2] in EuropeRecently Zika virus (ZIKV) also expanded into SouthernAmerica with reports of detection in Europe and USA [8]

Large surveillance and early warning systems commonlyapplied in European countries and around the world couldbenefit from a more sensitive and broader range screeningmethod Both mosquito pools and (sentinel) birds are com-mon targets of massive screening for arbovirus particularlyfor flaviviruses like WNV or USUV [6 9 10] Rapid virusidentification and quantification are crucial for accurate diag-nosis of ongoing infections treatment selection and follow-up as well as for selection and timely introduction of controlmeasures in outbreaks scenarios In this context highlyparallel detection technologies such asDNAmicroarrays aregaining importance [11ndash20]

Like RNA viruses in general flaviviruses are distin-guished by extensive genetic heterogeneity which implies

HindawiBioMed Research InternationalVolume 2017 Article ID 4248756 12 pageshttpsdoiorg10115520174248756

2 BioMed Research International

classification into subunits for example genotypes and lin-eages each with distinct epidemiological or clinical signif-icance This heterogeneity represents a major challenge inprimer and probe design for PCR and DNAmicroarray assaydevelopment

In the present study we have optimized a pan-Flavivirus-specific SYBR Green-based real-time reverse-transcriptionpolymerase chain reaction (RT-qPCR) [21] The amplifica-tion is subsequently complemented with hybridization ona low-density DNA microarray which exploits the geneticheterogeneity contained in the internal segment of the PCRamplicon thus allowing rapid identification of flavivirusesfrom clinical or field samples

2 Materials and Methods

21 Viral Samples and RNA Extraction The 26 Flavivirusstrains to be used as reference were collected and stored atminus80∘C until being processed as follows

African green monkey kidney (Vero) cells (Collectionof Cell Lines in Veterinary Medicine Friedrich-Loeffler-Institut Insel Riems Greifswald Germany) were infectedwith viruses in biosafety L3lowastlowast laboratory facilities and cellculture supernatants were collected and inactivated in BufferAVL (QIAGEN Hilden Germany) as previously described[22 23] The WNV strains lineage 1a-NY99 (ac AF196835)and Dakar lineage 1b-Kunjin (ac D00246) and lineage 2-Uganda 1937 (ac M12294) B956 (ac AY532665) Sarafend(ac AY688948) and goshawk Austria 36110 (2009) (acHM015884) were reused from our previous works [22ndash24]The strains USUV Germany 2011 (BH65) (ac HE599647)YFV 17D JEV TBEV Langat Malaysia 1956 (ac AF253419)and Murray Valley encephalitis virus (MVEV) were reusedfrom another work [25] USUV Austria 2001 (93901) andGermany 2011 (93901) were taken from [26] and JEVNakay-ama (ac EF571853) and TBEV Neudoerfl (ac U27495) from[27]

Additionally the following strains were obtained fromthe Health Protection Agency Salisbury United KingdomWNV lineage 2 MB 1957 SLEV YFV ldquoFrench Neurotroprdquoand LolIl (780) JEV Nakamura was kindly provided byA Mullbacher (John Curtin School of Medical ResearchCanberra Australia) RNA from ZIKV and DENV 1 2 3and 4 viruses was provided by P Despres (Flavivirus UnitInstitute Pasteur Paris France) while F Hufert and M Wei-dmann (Institute for Virology Gottingen Germany) kindlyprovided the viruses TBEV Absettarov 1951 (ac AF091005)TBEV Aina 1963 (ac AF091006) and TBEV Hypr 1953 (acU39292)

RNA was extracted with the RNeasy Mini Kit or theQIAamp Viral RNA Mini Kit (QIAGEN Hilden Germany)according to the manufacturerrsquos protocol A synthetic RNAwas used as internal extraction control (IC) [28] The NY99RNAwas used to create an external calibrator curve which inturnwas calibrated using the 51015840UTRWNV-specific RT-qPCR[22]

22 Selection of Primers andProbes Nearly 200 completeFlavi-virus genome sequences were obtained from the NCBINucleotide database by the end of 2010 Very similar se-quences (more than 98 identity)were excludedThe sequen-ces were aligned using CLUSTAL X [29] (accessed fromBioEdit software v7053) [30] and VectorNTI Advanced v10(Invitrogen Carlsbad CA USA) This alignment was manu-ally curated using both the nucleotide and the deduced aminoacid sequences and extended by adding partial sequences toreach a total of more than 400 so as to represent the NS5gene of all known species includingmost subtypes or lineages(a regularly updated version is available at Flavivirus GitHubsite (httpsgithubcomqPCR4virFlavivirus))

VisualOligoDeg (httpsgithubcomqPCR4virVisualO-liDeg) was used to facilitate visualization and selection ofappropriate oligonucleotides hybridizing to sequences of agivenTheNS5 regions (spanning nucleotides 9040 to 9305 ofAF196835) of the aligned sequences were imported into thisworkbook and were manually classified into major groups(MB mosquito borne TB tick borne and insect-only) virusgroups (JEVG YFVG TBEVG etc) species (WNV JEVYFV TBEV etc) and in some cases lineages (like WNV-1 orWNV-2 etc) or genotypes

Alongside species-specific RT-PCRs [31] broader genus-specific RT-PCR protocols have been also reported [1921 32 33] A genus-specific Taq-Man RT-PCR for Fla-vivirus detection was modified [21] with primer sequen-ces optimized using VisualOligoDeg It targets conservedflanking regions with an internal region with sufficient vari-ability to enable virus identification by sequencing the ampli-conThe original sequences were adapted to consistently am-plify most of the Flavivirus members resulting in degener-ate primers PFlav-fAAR (TACAACATGATGGGAAA-GAGAGAGAARAA from 9040 to 9068 of AF196835) andPFlavrKR (GTGTCCCAKCCRGCTGTGTCATC from posi-tions 9305 to 9283 of AF196835)

A total of 50 probes with a Tm around 55∘Cwere selectedA second set of sequences was also selected as a replace-ment in case of failure of first-set sequences The candidatesequences were submitted to the manufacturer for a final insilico evaluation of properties (homogenous hybridizationdiscriminatory potential etc) As a result all 84 sequenceswere found suitable for inclusion in the production of themicroarray (Table S01 in Supplementary Material availableonline at httpsdoiorg10115520174248756)

23 Detection andQuantificationUsing RT-qPCR Aone-stepSYBR Green-based RT-qPCR with melting curve analysiswas developed The QuantiTect SYBR Green RT-PCR Kit(QIAGEN Hilden Germany) was used following the manu-facturerrsquos instructions Briefly each primer (PFlav-fAAR andPFlav-rKR) was 51015840-biotinylated during the initial synthesis(Eurofins Genomics Ebersberg Germany) and used at afinal concentration of 08 120583M in a final reaction volume of25 120583L including 5120583L of RNA sample solution Real-time RT-qPCR was carried out on a CFX96 real-time PCR detectionsystem (Bio-Rad Laboratories Hercules USA) The thermalcycling profiles are presented in Table 1 Species-specific RT-qPCR were also used to determine the sensitivity of the

BioMed Research International 3

FlavivirusRT-qPCR Reference RNA samples includedWNV[22] USUV [26 34] and TBEV [35] In order to havecomparable Cq values across experiments when possiblewe set a separate fluorescence cut-off value for each target(PCR primer mix) in each 96-well plate run such that Cqasymp 28 for the control RNA WNV NY99 diluted 10minus4 Thiscontrol was previously calibrated using a synthetic RNA [22]In order to perform Flavivirus quantification an externalstandard curve consisting of four dilutions of RNA WNVNY99 10minus2 10minus3 10minus4 and 10minus5 was included in each runThese were equivalent to 4 times 104 4 times 103 4 times 102 and 40genome copies120583L of RNA solution or to 14 times 107 14 times 10614 times 105 and 14 times 104 copies per mL of homogenized samplerespectively To analyze the Flavivirus SYBR Green RT-PCRthe fluorescence was measured at step (6) of the standardprotocol (Table 1) Similarly for species-specific RT-qPCRfluorescence measurement was conducted at step (4)

24 Sequence Analysis Twenty 120583L of nonpurified PCR prod-uct was sent to Eurofins Genomics (Ebersberg Germany)for direct DNA sequencing by the Sanger dideoxy methodusing the amplification primers A BLAST (NCBI) search ofthe obtained sequences was usually sufficient to identify thevirus strain The (internal) amplicon of approximately 240 ntcontains sufficient phylogenetic information to reconstructphylogenetic trees and allows classification of more distantlyrelated and unknown strains and even new species

25 Microarray Analysis Flavivirus-specific oligonucleotideprobes selected using the VisualOligoDeg were spotted ontoa low-density microarray of the commercially availableArrayStrip (AS) platform (Alere Technologies GmbH JenaGermany)The 84 probes (spots 1ndash84 in S01 File) were spottedeither in triplicate (Chip Wildtech Virology-Mycob 01 from2011-01-13 assay ID-10610) [36] or in quintuplicate (ChipWildtech Virology 02 from 2012-09-10 assay ID-16050)The Alere Hybridization Kit was used following previouslypublished instructions [37 38] Briefly only positive RT-PCR reactions were routinely analyzed from which 1 120583L wasdirectly denatured in 100120583L of the hybridization buffer at95∘C for 5min and then placed for cooling at 4∘C for 5minin a thermocycler (Bio-Rad Laboratories) This solution wastransferred to the AS vessel (previously conditioned withwater and hybridization buffer) and incubated at 55∘C for1 h upon shaking at 550 rpm on a BioShake iQ heatableshaker (Quantifoil Instruments Jena Germany) The ASvessel was subsequently washed twice at 50∘C for 10minincubated with 100120583L of a peroxidase conjugate solutionat 30∘C for 10min and washed and incubated at roomtemperature with 100 120583L of the substrate solution (SeramunGrun Seramun Diagnostica GmbH Heidesee Germany) for5min Images of processedmicroarrays were saved in bitmap(bmp) format using the ArrayMate transmission Reader(Alere Technologies GmbH)

26 Microarray Data Processing and Flavivirus IdentificationThe web-based database Pionir The Experiment Navigatorof Partisan Array LIMS (Alere Technologies GmbH) was

Table 1 Thermal cycling profiles used in the Flavivirus RT-qPCR

Standard (45 h 25120583L) Fast (25 h 25 120583L or 10 120583L)(1) 50∘C for 30min(2) 95∘C for 15min(3) 95∘C for 15 s(4) 55∘C for 25 s + plate read(5) 72∘C for 25 s + plate read(6) 80∘C for 1 s + plate read(7) GOTO (3) 44 more times(8) 95∘C for 1min(9) Melting curve 68 to 88∘Cincrement 01∘C 1 s + plate read(10) 4∘C forever (optional)End

(1) 50∘C for 30min(2) 95∘C for 15min(3) 95∘C for 15 s(4) 55∘C for 20 s(5) 72∘C for 20 s + plate read(6) GOTO (3) 44 more times(7) 95∘C for 1min(8) Melting curve 68 to 86∘Cincrement 02∘C 1 s + plate readEnd

used for visualization and analysis of images and completeexperiments as well as for additional backup Alongside thePartisan IconoClust v36r0 software (Alere TechnologiesGmbH) was used for local analysis of microarray imagesgenerating for each spot of each picture the background-corrected signal intensities NI = 1 minus 119872BG with NI beingnormalized intensity 119872 average (mean) spot intensity andBG local background intensity Spot intensities are meas-ured as light transmission with119872 values ranging from 1 forcomplete transmission (background weak spots) to 0 forcomplete absorption (dark spots) Thus normalized signalintensities range between 0 and 1 Previous evaluation of thesignal to background ratio (SBR) in assays using the sametechnology has set the cut-off value for positive signals to01 [37] A custompython script (httpsgithubcomqPCR4virFlavivirusblobmastermicroarrayicono clust scriptsOnly-MeanSignalicrun) was embedded in this software to enablevisualization of individual hybridizations or batch analysis ofseries of experiments This script exports experimental datain different formats including a format suitable for importinto Orange software which allows visual programming andpython scripting for datamining and visualization (v276devinstalled 2014-06-06 from httporangebiolabsi)

Using Orange the visual program PanFlavExpStdSampl(httpsgithubcomqPCR4virFlavivirustreemastermicroar-rayorange) (Figure S01) was created which together withcustom modifications of some parts of Orange (httpsgithubcomqPCR4virorangecommit84f0a20e58b40b238f52319f-2017ae77df0dbf72) itself permits an interactive import ofthe experiments used as standards (known samples) forparallel analysis with unknown samples for classificationThis program calculates the distances between pairs of signalintensity patterns (two microarray experiments) which canserve as a measure of similarity between samples Severaldistance formulas are available interactively fromOrange andtheir meaning is explained elsewhere [39] These distancesare visualized in one of the widgets as heat-map-like graphics(Figure 5) in which the labels and the order of the samplescan be interactively selected from a group of preoptions Wehave added (directly modifying the source code of Orange(httpsgithubcomqPCR4virorangecommit21c3996c1712-baf8819e2050e7dccc31593cf2a0)) the option to reorganizethe heat map showing the selected sample at the top followed


by the most similar samples by mouse-clicking the respectivecell This graphic can also show the samples organized ina tree to reveal clustering Another Orange widget uses thedistances to construct a tree (Figure 4) in which a cut-offcan be interactively selected andor groups defined to makea report of the proposed classification

27 Field Samples After development initial evaluationand optimization the new assay was further evaluated bytesting RNA extracts from mosquito pools from GreeceMosquito trapping was performed within the framework of asurveillance program which was implemented in the regionof Central Macedonia during the 2012 arbovirus transmis-sion period The aims of this program were to molecularlycharacterize WNV and assess population dynamics of themajor arbovirus vector species to timely notify public healthauthorities on increased risk [40] Female adult mosquitoeswere collected at dry ice-baited CDC mosquito traps whichwere set in areas with previous indications of high arbovirusprevalence [40]

Identified mosquitoes (50 individuals of the same genus)were placed in 2mL microcentrifuge tubes with two 4mmsterile glass balls and 1mL of phosphate-buffered saline (PBS)containing 1 of heat-inactivated fetal bovine serum (FBSSigma-Aldrich Steinheim Germany) Disruption was per-formed for 30 s at speed 40 using a RiboLyser homogenizer(Hybaid Ltd Teddington UK) The homogenates were cen-trifuged (16000timesg 5min at 4∘C) and 150 120583L of supernatantfrom eachmosquito pool underwent RNA isolation based onthe previously described RNA extraction method

In total 340 mosquito pools were tested with the Fla-vivirus RT-qPCR protocol Out of them 180 pools wererepresented by Culex mosquitoes 75 were comprised byAedes and 85 were comprised by Anophelesmosquitoes

3 Results

Following preliminary observations that the sensitivity andefficiency of amplification with the primers from [21] werenot homogeneous for different flaviviruses we decided todesign a modified set of degenerate primers Using Visu-alOligoDeg we selected primers for a modified RT-qPCRfor Flavivirus detection and quantification In the initialexperiments we compared the newprocedurewith publishedRT-qPCRs using reference viruses All 26 Flavivirus referencestrains described in Section 21 were tested (Figure 1)

The optimized Flavivirus RT-qPCR was also comparedin terms of detection limit with species-specific RT-qPCRsfor WNV USUV and TBEV by testing fresh RNA solutions(from supernatants of infected cells) in a series of end-pointdilution experiments (Figures S02 and S03)

Subsequently we conducted melting curve analysiswhich can provide useful information to determine positivityand sequence differences (as shown in Figure 2) butwhich haslimited value for identification A well-defined peak between79 and 84∘C is a strong indication of positivity anddifferencesin Tm indicate sequence differences possibly representingdifferent species or lineages The results from end-point

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1000

1500

2000

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dilution experiments and the calculations using the externallycalibrated standard curve suggest that for most membersof Flavivirus genus this RT-qPCR assay is highly sensitivecapable of detecting a few viral RNA copies per reactionHowever this also means that cross-sample contaminationand DNA carryover are a major concern Therefore it iscrucial to organize the laboratory work accordingly andinclude sufficient controls to validate the results of eachexperiment

To evaluate the accuracy and performance of the devel-oped RT-qPCR assay we successfully participated in fiveinternational ring trials for quality assessment of nucleic acidamplification tests that is ANSES 2013 (melting curves ofthe samples shown in Figure 2) Quality Control for Molec-ular Diagnostics or QCMD-2010QCMD-2011 [41] QCMD-2012 and QCMD-2013 (results summarized in Table S02)(httpwwwqcmdorg) These results complemented theinformation obtained from WNV-specific RT-qPCRs Both


Table 2 Screening of mosquito pools collected in Greece Review of the screening of the 340 mosquito pools (50 mosquitoes each) collectedin Greece All RT-qPCR positive and 6 RT-qPCR negative pools were tested further by microarray and direct sequencing All other poolstested showed a Cq gt 38 and Tm lt 78∘C

Mosquito pool Cq Tm (∘C) Microarray DNA sequencing resultMPGr01 373 814 WNV2 Austria Nea Santa-Greece-2010 HQ537483MPGr02 316 816 WNV2 Austria Nea Santa-Greece-2010 HQ537483MPGr03 215 817 WNV2 Austria Nea Santa-Greece-2010 HQ537483MPGr04 325 819 WNV2 Austria Nea Santa-Greece-2010 (with 1 mutation)MPGr05 385 816 WNV2 Austria NegativeMPGr06 356 804 Marisma mosquito virus (93 identity)MPGr07 396 810 Culex Flav New Mosq Culex Flavivirus (78 identity)MPGr08 398 794 Negative NegativeMPGr09 414 830 Negative NegativeMPGr10 386 812 Negative Negative (residual sequence Salmonella sp)MPGr11 401 824 Negative NegativeMPGr12 406 792 Negative NegativeMPGr13 396 807 Negative Negative (residual sequence Pseudomonas sp)

WNV and non-WNV Flavivirus strains were quantified usingthe Flavivirus RT-qPCRwith theWNV calibration curve andwere subsequently identified by microarray analysis

The combined RT-qPCRmicroarray procedure was ap-plied on the 26 reference virus strains and RT-qPCR positivefield samples of diverse origin in more than 300 hybridiza-tion experiments Each analyzed reference virus produceda specific hybridization pattern that allowed discriminationMost flaviviruses could be identified at the level of speciesgenotype or even strain following comparison of theirhybridization patterns with those of reference samples Acompilation of four Flavivirus isolates each examined attwo different RNA dilutions is shown in Figure 3 Thisfigure reveals an important distinction of this microarrayplatform from well-known glass-slide arrays used for geneexpression studies meaning that the developed microarrayis optimized to detect genetic (sequence) variations ratherthan the concentration or relative quantity of ampliconsThus the present microarray signal intensity values are usedsolely for identification or classification while quantificationis performed in the preceding RT-qPCR step As shownin Figure 3 the hybridization patterns are not significantlyaffected by quantitative variations in the viral load of thesample but qualitative changes are readily visible whendifferent strains of the same virus species are examined

In some cases virus identification is already possible byvisually comparing the signal patterns of the bar diagrams(eg those in Figure 3) However given the complexity ofthe signals it was necessary to include a computer-basedsolution for data processing Orange was used to create avisual program (Figure S01) to import the raw data from theIcono Clust software to define a set of experimental standardsand identify viral samples by clustering (Figure 4) Theprocedure also permits visualization of mixed of Flavivirusinfections in heat-map-like graphics (Figure 5) This capa-bility represents another major advantage of the microarraycompared to direct sequencing Mixes of RNA from closely

related Flavivirus strains were tested to explore the possibilityof detection of coinfections in a given sample or detectionof the presence of more than one virus in tested pools Wehave selected USUV and WNV-1a and WNV-2 (NY99 andUg37) viruses to show that it is possible to unambiguouslydetect each component in a mix even of related viruses suchas lineages 1 and 2 of the WNV (Figures 5(b) and 5(c))

The Flavivirus RT-qPCR screening was conducted onthe 340 mosquito pools from Greece PCR products of 13mosquito pools (including all positive specimens as well assix RT-qPCR-negative pools) underwent microarray analysis(Table 2) which revealed the presence of WNV lineage2 sequences similar to the Austria strain in five of them(MPGr01-MPGr05 for sim15 of the total number of poolstested) Culex mosquitoes comprised all five WNV-positivepools One pool yielded a strong positive result (Cq 215)two were of mediums (Cq ndash 316 and 325) and two were ofweak concentrations (Cq 373 and 385) Sequencing of thesefive amplicons revealed identity to WNV isolates goshawk-Hungary04 DQ116961 Nea Santa-Greece-2010 HQ537483[42] and Italy 2013 KF647248 in four cases while the fifth(the one with the highest Cq value) was not sequenceable

The microarray also detected weak signals of insect-specific flaviviruses (ISFVs) in two of the pools tested DNAsequencing revealed the presence of sequences with similar-ities to the isolate HU452807 of Marisma mosquito virus(JN603190 93 nucleotide identity) in one Aedes sp pool(MPGr06) Additionally a presumably new mosquito Fla-vivirus (with only 78 identitywithGQ165809 theNakiwogovirus strain from Uganda) was also detected in an Anophelessp pool (MPGr07) Flavivirus RT-qPCRmicroarray screen-ing of the present panel was completed in less than threeworking days

Additionally other specimens were analyzed includingorgans (such as brain heart liver lung kidney and spleen)and blood from falcons [24] pheasants blackbird greatgrey owl common kingfisher and nearly 70 other avian


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1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

WNV2Ug x 10-4

WNV2Ug x 10-4

WNV2Ug x 10-1

WNV2Ug x 10-1

(b)

000010020030040050060070080090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

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eng

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219

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gf1

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illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

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2f9

3f9

4f9

5f9

6f9

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23

JE2

j38

JEj

33JE

j34

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35

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36

JEjj

37

JEVG

Igf

39JE

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f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

MVV15 x 10-3

MVV15 x 10-3

MVV15 x 10-1

MVV15 x 10-1

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(c)

Figure 3 Continued


aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

TBLang18x10-3

TBLang18x10-3

TBLang18x10-1

TBLang18x10-1

000010020030040050060070080090

(d)

Figure 3 Hybridization patterns for different dilutions of viral samples Cell culture supernatants (a)West Nile virus lineage 1a strain NY99(WNV1aNY) red bars sample diluted 10minus1 and blue bars diluted 10minus4 (b) West Nile virus lineage 2 strain Uganda 1937 (WNV2Ug) redbars 10minus1 and blue bars 10minus4 (c) Murray Valley encephalitis virus (MVEV) red bars 10minus1 and blue bars 10minus3 (d) Tick-borne encephalitisvirus strain Langaat (TBLang) red bars 10minus1 and blue bars 10minus3 These data show that hybridization patterns are very sensitive to variationin the viral sequence while remaining stable in a broad concentration range of viral RNA

species [6] as well as from mice camels horses donkeyscattle humans mosquitoes and ticks Only horse sampleshave caused artifacts in the RT-qPCR showing low nonspe-cific signals (Cq gt 33) thus effectively lowering the detectionlimit of RT-qPCR alone in horse samples to over 50 copies perreaction (data not shown)

4 Discussion

By designing the present combined RT-qPCRmicroarrayassay for detection quantification and identification offlaviviruses a number of methodological problems havebeen solved Using VisualOligoDeg we selected primersand probes for the newly developed assay The ability of arelatively accurate quantification during the RT-qPCR phaseof the assay is one of its major advantages Technically thecombined RT-qPCRmicroarray assay is easy to handle asonly standard experience with real-time PCR and ELISA-like tests is required We regularly achieved complete testingof samples in one working day from RNA extraction tofinal visualization of the tree and interactive heat-map-likegraphics

The present assay permits classification andor identi-fication up to the (sub)lineage level avoiding in most casesthe need for sequencing It has been shown to be as sensitiveas species-specific RT-qPCRs and suitable for broad-rangeFlavivirus screening as well as a confirmatory assay in bothlaboratory and field samplesThe present study has also dem-onstrated that the assay can be efficiently used in arbovirussurveillance programs for rapid screening and discrimina-tion of flaviviruses for example in mosquito or animal spec-imens In areas where numerous arbovirus strains of different

virulence cocirculate such as in Greece and other Euro-pean countries molecular identification of the circulatingviruses is a necessity [10] Especially for flaviviral zoonosesphylogeography and identification of virulent strains are ofutmost importance The assay is also capable of detectinginsect-specific flaviviruses A report on the presence ofISFVs in Culex mosquitoes of Central Macedonia-Greecealready exists [43] Application of RT-qPCRmicroarray test-ing revealed the presence of a virus strain with sequencesimilarities to Marisma mosquito virus as well as a presum-ably novel mosquito Flavivirus sequence in the same areaof Greece The detection of these ISFVs via the combinedRT-qPCRmicroarray protocol extends our knowledge on thepresence of mosquito flaviviruses in GreeceThe broad rangeof flaviviruses that are being tested simultaneously in thisassay in combination with its convenience and theminimumtime required for obtaining the results makes it a useful toolthat potentially can be applied widely for surveillance andepidemiological surveys

While our assay has been proven to be suitable for diag-nostic and research laboratories it represents an open systemthat can be further improved In case of newly emergingpathogenic and genetically distinct strains primer sequencescould be further optimized using newly available genomesequences andor improving probes of those species that werenot in the focus of the present study (possibly DENV 4)The number of probes on the microarray is currently at 84but could be increased to 500 using the present technologySince we relied on the specifications of the commercialhybridization kit we did not perform extensive optimizationof the incubation times and temperature and will evaluate the


054 040 027 013 000

054 040 027 013 000

NTCx316H12x318G01NTCQCMD2013x279H02WNV1aNY99-8x311E12x318E01WNV1a-NY9911copx210A03RingTest201101WNV1aNY991000c_mlQCMD2013x279A02WNV1aIs981ANSES2013x280A02WNVNY99x10-4x280A03WNV1aDakar-91x10-3x321D01x322D01

WNV1aNY99x10-4x273B01WNVDakarWNVDakarWNVDakarWNV1aNY99WNVNY-4x229A12

WNV2Aus08ANSES2013x280B02WNV2Aus08ANSES2013x280H03WNVHeja10+6c_mlQCMD2013x279C02WNV2AustriaWNVHeja12000 copRTStWNV1104WNV2 100000c_mlQCMD2013x279D02

WNVMBWNVMBWNV2Ug37x10-4x273A01 WNVUg x 10-4WNVUg x 10-1

WNVUgWNVUg37100000c01-B266072910610-2WNV2Sarafend-62x10-2x321E02x322E02WNV2SarafWNV2SarafMVV-1514x10-3x321E01x322E01

MVV15 x 10-1

MVVMVVUsutuIta12ANSES2013x280D02Usutux275C01xH02Usutu-36x10-2x321D02x322D02Usutu33 x 10-3

Usutu 36Usutu x10-5x273D01

UsutuUsutu x10-5 Exp25102-G266073010610_7JEVNakayamaANSES2013x280G02JEVNak5x10-3mplexx279G03x276C06JENYJENMJENmJEVNakayamax10-4x273C01JENYTBEVHyprANSES2013x280F02TBEVAbsetTBEVHyprTBEVHyprx10-3x279A01TBEVAinaTBEVAx10-3x279B01TBEVAinaTBEVHypr-3910-4x321F01x322F01TBEVNeud38x10-3mplexx279C03x276G06TBEVAbsetTBEVHyprTBEVAinaLoupIl-277x10-4x321A01x322A01LoupIlLoupIl

TBEVLangatTBEVLangat

TBEVLangat18x10-3TBEVLangat19x10-1WNV1bKunjiWNV1bKunjiWNV1bKunjiSLEVWeidm-297x311E03xx318D01SLEV-297x10-3x321G01x322G01SLEV29x10-3SLEV29x10-1SLEV7x10-2mplexx279A03x276F06

SLEV7x10-2mplexx279B03x276F07

SLEVYFV17D191x10-2x321B01x322B01YFVFrench-222x10-2x321C01x322C01YFVFN21 x10-3YFVFN21 x10-1

YFV17DYFV2x10-2mplexx279E03x276D06

YFVFN

WNV1aNY99x10-4 55∘C

WNVWNVNY99(simc120583l) QCMD-112012EX25101-E266073110610_5

WNV2Ug x10-4 55∘C

WNVUg x10-4 50∘C

MVV10-3 55∘C

MVV x 10-3 50∘C

Usutu10-3 55∘C

Usutu x 10-3 50∘C

TBEVLangat x 10-3 50∘C


SLEV x 10-3 55∘C

SLEV x10-3 50∘C

YFVFN x10-3 55∘C

YFVFN X10-3 50∘C

Figure 4 Cluster analysis of tested samples Output of the tree widget inOrange software A cluster analysis was conducted using the distancesbetween the hybridization patterns of experimentally tested samples This is the preferred method to visualize the identification of viruses insamples that contain only one Flavivirus

possibility of using future versions of that kit [44] A limita-tion of the present assay is the requirement of experimentalhybridizations of known samples which does not allow directclassification of unknowns Nevertheless the methodology isrobust enough to allow the use of theoretical hybridizationpatterns or those obtained in other laboratories which couldbe a solutionwhen a particular virus reference is not availableIt should be emphasized that the tree constructed by the

Orange tree widget using the distances between hybridizationpatterns is not a phylogenetic tree The graphics just help togroup samples according to sequence-based relatedness andthereby facilitates their identification The Orange softwarealso provides possibilities for classification using machine-learning methods which have yet to be explored but theyhave the potential to significantly improve the accuracy of thefinal report


JEVNakayamax10-4x273C01JENy

JENmJENm

JEVNak5x10-3mplexxx279G03x276C06JEVNakayamaANSES2013x280G02

2013D1001-Cchip2013B0201-Fchip

2605H1203-Hchip2013G0301-Achip2535B0502-Bchip

JENy2013C0901-Bchip

JENy2013C0901-Bchip

2535A0901-GchipWNV1aNY99-8x311E12x318E01

WNV1aIs981ANSES2013x280A02WNV1aNY99x10-4x280A03

WNV1aNY991000c_mlQCMD2013x279A02WNV1aNY9911copx210A03 Ring Test201101

WNV1aNYx10-4x273B01WNVHeja12000copRTstWNV1104

WNVDakarWNV1aDakar-91x10-3x321D01x322D01

WNV1NY-4x229A12WNV1aNY99

JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

A09

01-

Gch

ipW

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1W

NV

1aIs

981

AN

SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99

Dissimilarity000 070

WN

V1

aNY9

9x1

0-4

55∘ C


(a)

mxUsutux10-4WNV1aNY9910-4x280E01

WNV1aIs981ANSES2013x280A02

WNV1aNY99x10-4x280A03

WNV1aNY9911copx210A03RingTest201101

WNV1aNY991000c_mlQCMD2013x279A02

WNV1aNY99-8x311E12x318E01

WNV1aDakar

WNV1aDakar-91x10-3x321D01x322D01

WNV1aNY-4 x229A12

Usutux275C01xH02

mixUsutux10-4WNV2Ug37x10-4WNV1a

UsutuIta12ANSES2013x280D02

Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

x10

-4W

NV

1aN

Y99

10-4

x28

0E01

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

E01

WN

V1a

Dak

ar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1a

NY-

4 x

229

A12

Usu

tux

275C

01x

H02

Usu

tuIt

a12

AN

SES2

013

x280

D02

Ustu

36

Usu

tux

10-5

x27

3D01

Usu

tu-3

6x10

-2x

321D

02x

322D

02

Usu

tu3

3 x1

0-3

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a


WN

V1

aNY9

9x1

0-4

55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued


mixUsutux10-4WNV2Ug37x10-4WNV1aNY99x10-4mixWNV2Ug37x10-4WNV1aNY99x10-4x280G01

mixUsutux10-5WNV2Ug37x10-4x280F01WNV2Ug3710000c_mlQCMD2013x279D02

WNV2Ug x10-4WNV1aNY-4x2999A12

WNVHeja12000copRTstWNV1104WNV2Austria

WNV2Ug37x10-4x273A01WNV2Ug x 10-1

WNV2SarafUsutu

WNV2Sarafend-62x10-2x321E02x322E02

Usutu x10-5 Exp25102-G266073010610_7WNVDakar

WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01

Mix Usutu x10-5+WNVNYx10-4Mix Usutux10-4WNV1aNY99x10-4x280E01

WNV2Aus08ANSES2013x280F03WNV2Aus08ANSES2013x280D03WNV2Aus08ANSES2013x280B02

WNV1aNY99x10-4x273B01WNV2Saraf

WNV1aDakar-91x10-3x321D01x322D01WNVHeja10+6c_ml-QCMD2013x279C02

WNV1a-NY9911copx210A03RingTest201101WNV2Aus08ANSES2013x280A03

WNV1aIs981ANSES2013x280A02WNV1aNY99-8x311E12x318E01

Usutu36WNV2Ug37100000c01-B266072910610_2

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

mix

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

x280

G01

mix

Usu

tux

10-5

WN

V2

Ug3

7x1

0-4

x280

F01

WN

V2

Ug3

710

000c

_mlQ

CMD

2013

x279

D02

WN

V2

Ug

x10-

4W

NV

1aN

Y-4

x299

9A

12W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

V2

Austr

ia

WN

V2

Ug3

7x1

0-4

x273

A01

WN

V2

Ug

x 10

-1W

NV

2Sa

raf

Usu

tuW

NV

2Sa

rafe

nd-6

2x1

0-2

x321

E02

x322

E02

Usu

tu x

10-5

Exp

251

02-G

266

0730

1061

0_7

WN

VD

akar

WN

VM

BW

NV

MB

WN

V1a

NY9

9M

VV

-15

14x1

0-3

x321

E01

x322

E01

Mix

Usu

tu x

10-5

+WN

VN

Yx10

-4M

ix U

sutu

x10

-4W

NV

1aN

Y99

x10-

4x2

80E0

1W

NV

2Au

s08

AN

SES2

013

x280

F03

WN

V2

Aus0

8A

NSE

S201

3x2

80D

03W

NV

2Au

s08

AN

SES2

013

x280

B02

WN

V1a

NY9

9x1

0-4

x273

B01

WN

V2

Sara

f

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

VH

eja1

0+6c

_ml-Q

CMD

2013

x279

C02

WN

V1a

-NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V2

Aus0

8A

NSE

S201

3x2

80A

03

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02W

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1U

sutu

36

WN

V2

Ug3

710

0000

c01-

B26

6072

9106

10_2

Usu

tu x10

-350∘ C

WN

V2

Ug

x10

-455∘ C

WN

V1

aNY9

9x1

0-4

55∘ C

WN

V2

Ug

x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C

WNV1aNY99 x10-4 55∘C

WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)

Figure 5 Presentation of experimental results processed by theOrangersquos heat-map-like widget showing an all-versus-all sample comparisonCalculated distances between intensity patterns of each pair ofmicroarray results obtained fromhybridization of sample amplicons are shownwith each cell representing the comparison of two microarray experiments The dissimilarity color scale is shown at the top Columns androws are organized in the same order making the diagonal an ldquoidentityrdquo sample comparisonWhen one sample is selected bymouse-clickingthe map is immediately reorganized to show this sample in the upper row and left column followed by the most similar samples (a) Theselection of the ldquoJEVNakayamax10-4x273C01rdquo sample reveals a good separation of JEV samples from all other viral species (b) The sameheat map after selecting a sample containing a mix of diluted USUV and WNV1a RNA which is easily recognized by a framed mosaic-likemap (c) A more complex mix including USUV WNV1 and WNV-2 RNA was selected showing a more complex pattern that still permitsthe identification of the components (which can be additionally confirmed by the successive selection of each of the three standard WNV-1WNV-2 and USUV to check that this mix clusters together with each of them)

Compared to the diagnostic assays for Flavivirus detec-tion published so far our procedure is distinguished by itshigh degree of parallelity in detection of a wide range ofvirus species strains and their variations which cannotbe achieved through ldquoone-dimensionalrdquo RT-PCR assays [2132 45] Previously published microarray or diagnostic chipapproaches [20] lack the ease of operation of the ArrayStripplatform used here and cover only part of the range of virusesthat we can identify [12 14 19 46]

5 Conclusion

We have developed a combined RT-qPCR-microarray assayfor high-throughput screening and identification of fla-viviruses including mixed infections of different species orstrains Our experience in analyzing field samples (fromticks and mosquito vectors and from human and animal

samples of different sources) shows that the assay allowsrapid and highly sensitive screening and identification ofFlavivirus strains within one day The assay has helped toovercome limitations in virological diagnosis due to lackof specificity or sensitivity in conventional and real-timeRT-PCR protocols Even if direct sequencing is used asgenotyping tool the developed microarray can be a used asa rapid complementary test to detect mixtures of differentFlavivirus strainsThe good performance of the assaywas alsoconfirmed by correctly quantifying and identifyingmembersof the Flavivirus genus in samples from international ringtrials for quality assessment of nucleic acid amplification tests

Disclosure

Konrad Sachsersquos present address is Department ofBioinformatics for High-Throughput Analysis Faculty of


Mathematics and Computer Science Friedrich-Schiller-Uni-versitat Jena Germany

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Alexandra Chaskopoulou (European Biological Control Lab-oratory USDA-ARS Thessaloniki Greece) is acknowledgedfor providing identified mosquitoes

References

[1] E Gould and T Solomon ldquoPathogenic flavivirusesrdquoThe Lancetvol 371 no 9611 pp 500ndash509 2008

[2] M G Guzman and E Harris ldquoDenguerdquo The Lancet vol 385no 9966 pp 453ndash465 2015

[3] T P Monath and P F C Vasconcelos ldquoYellow feverrdquo Journal ofClinical Virology vol 64 pp 160ndash173 2015

[4] R S Lanciotti J T Roehrig V Deubel et al ldquoOrigin of theWest Nile virus responsible for an outbreak of encephalitis inthe Northeastern United Statesrdquo Science vol 286 no 5448 pp2333ndash2337 1999

[5] J N Hanna S A Ritchie D A Phillips et al ldquoJapaneseencephalitis in north Queensland Australia 1998rdquo MedicalJournal of Australia vol 170 no 11 pp 533ndash536 1999

[6] U Ziegler H Jost K Muller et al ldquoEpidemic spread of usutuvirus in Southwest Germany in 2011 to 2013 and monitoring ofwild birds for Usutu and West Nile virusesrdquo Vector-Borne andZoonotic Diseases vol 15 no 8 pp 481ndash488 2015

[7] M Marcantonio A Rizzoli M Metz et al ldquoIdentifying theenvironmental conditions favouring West Nile Virus outbreaksin Europerdquo PLoS ONE vol 10 no 3 Article ID e0121158 2015

[8] A S Fauci and D M Morens ldquoZika virus in the americasmdashyetanother arbovirus threatrdquoNewEngland Journal ofMedicine vol374 no 7 pp 601ndash604 2016

[9] S C Chaintoutis C I Dovas M Papanastassopoulou et alldquoEvaluation of a West Nile virus surveillance and early warningsystem in Greece based on domestic pigeonsrdquo ComparativeImmunology Microbiology and Infectious Diseases vol 37 no2 pp 131ndash141 2014

[10] A Chaskopoulou C I Dovas S C Chaintoutis J KashefiP Koehler and M Papanastassopoulou ldquoDetection and earlywarning of West Nile virus circulation in Central MacedoniaGreece using sentinel chickens and mosquitoesrdquo Vector-Borneand Zoonotic Diseases vol 13 no 10 pp 723ndash732 2013

[11] L G Glushakova A Bradley K M Bradley et al ldquoHigh-throughput multiplexed xMAP Luminex array panel for detec-tion of twenty two medically important mosquito-bornearboviruses based on innovations in synthetic biologyrdquo Journalof Virological Methods vol 214 pp 60ndash74 2015

[12] J J L Tan M Capozzoli M Sato et al ldquoAn integrated lab-on-chip for rapid identification and simultaneous differentiation oftropical pathogensrdquo PLoS Neglected Tropical Diseases vol 8 no7 Article ID e3043 2014

[13] N D Grubaugh L N Petz V R Melanson et al ldquoEvaluationof a field-portable DNA microarray platform and nucleic acid

amplification strategies for the detection of arboviruses arthro-pods and bloodmealsrdquo American Journal of Tropical Medicineand Hygiene vol 88 no 2 pp 245ndash253 2013

[14] N D Grubaugh S S McMenamy M J Turell and J SLee ldquoMulti-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarrayrdquo PLoSNeglected Tropical Diseases vol 7 no 8 Article ID e2349 2013

[15] D D Singh and A Jain ldquoMultipurpose instantaneous microar-ray detection of acute encephalitis causing viruses and theirexpression profilesrdquo Current Microbiology vol 65 no 3 pp290ndash303 2012

[16] J Teo P D Pietro F S Biagio et al ldquoVereFlu an integratedmultiplex RT-PCR andmicroarray assay for rapid detection andidentification of human influenza A and B viruses using lab-on-chip technologyrdquo Archives of Virology vol 156 no 8 pp 1371ndash1378 2011

[17] X Kang C Qin Y Li et al ldquoImprovement of the specificity of apan-viral microarray by using genus-specific oligonucleotidesand reduction of interference by host genomesrdquo Journal ofMedical Virology vol 83 no 9 pp 1624ndash1630 2011

[18] X-P Kang Y-Q Li Q-G Sun H Liu Q-Y Zhu and Y-H Yang ldquoDevelopment of a consensus microarray method foridentification of some highly pathogenic virusesrdquo Journal ofMedical Virology vol 81 no 11 pp 1945ndash1950 2009

[19] H Nordstrom K I Falk G Lindegren et al ldquoDNAmicroarraytechnique for detection and identification of seven flavivirusespathogenic for manrdquo Journal of Medical Virology vol 77 no 4pp 528ndash540 2005

[20] J Korimbocus N Scaramozzino B Lacroix J M Crance DGarin and G Vernet ldquoDNA probe array for the simultaneousidentification of herpesviruses enteroviruses and flavivirusesrdquoJournal of Clinical Microbiology vol 43 no 8 pp 3779ndash37872005

[21] D-Y Chao B S Davis and G-J J Chang ldquoDevelopmentof multiplex real-time reverse transcriptase PCR assays fordetecting eightmedically important flaviviruses inmosquitoesrdquoJournal of ClinicalMicrobiology vol 45 no 2 pp 584ndash589 2007

[22] M Eiden A Vina-Rodriguez B Hoffmann U Ziegler andM H Groschup ldquoTwo new real-time quantitative reversetranscription polymerase chain reaction assays with uniquetarget sites for the specific and sensitive detection of lineages1 and 2West Nile virus strainsrdquo Journal of Veterinary DiagnosticInvestigation vol 22 no 5 pp 748ndash753 2010

[23] K Schmidt M Keller B L Bader et al ldquoIntegrins modulatethe infection efficiency of West Nile virus into cellsrdquo Journal ofGeneral Virology vol 94 no 8 pp 1723ndash1733 2013

[24] U Ziegler J Angenvoort D Fischer et al ldquoPathogenesis ofWest Nile virus lineage 1 and 2 in experimentally infected largefalconsrdquo Veterinary Microbiology vol 161 no 3-4 pp 263ndash2732013

[25] N Becker H Jost U Ziegler et al ldquoEpizootic emergence ofUsutu virus in wild and captive birds in Germanyrdquo PLoS ONEvol 7 no 2 Article ID e32604 2012

[26] H Jost A Bialonski D Maus et al ldquoIsolation of usutu virus inGermanyrdquo American Journal of Tropical Medicine and Hygienevol 85 no 3 pp 551ndash553 2011

[27] U Ziegler A Skrypnyk M Keller et al ldquoWest nile virusantibody prevalence in horses of Ukrainerdquo Viruses vol 5 no10 pp 2469ndash2482 2013

[28] B Hoffmann K Depner H Schirrmeier and M Beer ldquoAuniversal heterologous internal control system for duplex real-timeRT-PCR assays used in a detection system for pestivirusesrdquo


Journal of Virological Methods vol 136 no 1-2 pp 200ndash2092006

[29] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[30] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[31] C Domingo P Patel S Linke K Achazi and M NiedrigldquoMolecular diagnosis of flavivirusesrdquo Future Virology vol 6 no9 pp 1059ndash1074 2011

[32] P Patel O Landt M Kaiser et al ldquoDevelopment of one-stepquantitative reverse transcription PCR for the rapid detectionof flavivirusesrdquo Virology Journal vol 10 article no 58 2013

[33] M P Sanchez-Seco D Rosario C Domingo et al ldquoGenericRT-nested-PCR for detection of flaviviruses using degeneratedprimers and internal control followed by sequencing for specificidentificationrdquo Journal of Virological Methods vol 126 no 1-2pp 101ndash109 2005

[34] F Cavrini M E Della Pepa P Gaibani et al ldquoA rapid andspecific real-time RT-PCR assay to identify Usutu virus inhuman plasma serum and cerebrospinal fluidrdquo Journal ofClinical Virology vol 50 no 3 pp 221ndash223 2011

[35] M Schwaiger and P Cassinotti ldquoDevelopment of a quantitativereal-time RT-PCR assay with internal control for the laboratorydetection of tick borne encephalitis virus (TBEV)RNArdquo Journalof Clinical Virology vol 27 no 2 pp 136ndash145 2003

[36] A Ruettger J Nieter A Skrypnyk et al ldquoRapid spoligotypingof Mycobacterium tuberculosis complex bacteria by use of amicroarray system with automatic data processing and assign-mentrdquo Journal of Clinical Microbiology vol 50 no 7 pp 2492ndash2495 2012

[37] C Schnee S Schulsse H Hotzel et al ldquoA novel rapid dnamicroarray assay enables identification of 37 mycoplasmaspecies and highlights multiple mycoplasma infectionsrdquo PLoSONE vol 7 no 3 Article ID e33237 2012

[38] C Schnee and K Sachse ldquoDNA microarray-based detection ofmultiple pathogens Mycoplasma spp and Chlamydia spprdquo inVeterinary Infection Biology Molecular Diagnostics and High-Throughput Strategies vol 1247 ofMethods inMolecular Biologypp 193ndash208 Springer 2015

[39] A Urisman K F Fischer C Y Chiu et al ldquoE-Predict a com-putational strategy for species identification based on observedDNA microarray hybridization patternsrdquo Genome Biology vol6 no 9 article R78 2005

[40] S C Chaintoutis A Chaskopoulou T Chassalevris P GKoehler M Papanastassopoulou and C I Dovas ldquoWest nilevirus lineage 2 strain in Greece 2012rdquo Emerging InfectiousDiseases vol 19 no 5 pp 827ndash829 2013

[41] S Linke W G MacKay C Scott P Wallace and M NiedrigldquoSecond external quality assessment of themolecular diagnosticof West Nile virus are there improvements towards the detec-tion of WNVrdquo Journal of Clinical Virology vol 52 no 3 pp257ndash260 2011

[42] A Papa T Bakonyi K Xanthopoulou A Vazquez A Tenorioand N Nowotny ldquoGenetic characterization of west nile viruslineage 2 Greece 2010rdquo Emerging Infectious Diseases vol 17 no5 pp 920ndash922 2011

[43] A Papa E Papadopoulou S Kalaitzopoulou K Tsioka andS Mourelatos ldquoDetection of West Nile virus and insect-specific flavivirus RNA in Culex mosquitoes central Mace-donia Greecerdquo Transactions of the Royal Society of TropicalMedicine and Hygiene vol 108 no 9 pp 555ndash559 2014

[44] I V Jani B Meggi A Vubil et al ldquoEvaluation of the whole-blood alere q nat point-of-care RNA assay for HIV-1 viral loadmonitoring in a primary health care setting in MozambiquerdquoJournal of Clinical Microbiology vol 54 no 8 pp 2104ndash21082016

[45] M W Gaunt and E A Gould ldquoRapid subgroup identificationof the flaviviruses using degenerate primer E-gene RT-PCR andsite specific restriction enzyme analysisrdquo Journal of VirologicalMethods vol 128 no 1-2 pp 113ndash127 2005

[46] A J Foord V Boyd J R White D T Williams A Collingand H G Heine ldquoFlavivirus detection and differentiation bya microsphere array assayrdquo Journal of Virological Methods vol203 pp 65ndash72 2014

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


classification into subunits for example genotypes and lin-eages each with distinct epidemiological or clinical signif-icance This heterogeneity represents a major challenge inprimer and probe design for PCR and DNAmicroarray assaydevelopment

In the present study we have optimized a pan-Flavivirus-specific SYBR Green-based real-time reverse-transcriptionpolymerase chain reaction (RT-qPCR) [21] The amplifica-tion is subsequently complemented with hybridization ona low-density DNA microarray which exploits the geneticheterogeneity contained in the internal segment of the PCRamplicon thus allowing rapid identification of flavivirusesfrom clinical or field samples

2 Materials and Methods

21 Viral Samples and RNA Extraction The 26 Flavivirusstrains to be used as reference were collected and stored atminus80∘C until being processed as follows

African green monkey kidney (Vero) cells (Collectionof Cell Lines in Veterinary Medicine Friedrich-Loeffler-Institut Insel Riems Greifswald Germany) were infectedwith viruses in biosafety L3lowastlowast laboratory facilities and cellculture supernatants were collected and inactivated in BufferAVL (QIAGEN Hilden Germany) as previously described[22 23] The WNV strains lineage 1a-NY99 (ac AF196835)and Dakar lineage 1b-Kunjin (ac D00246) and lineage 2-Uganda 1937 (ac M12294) B956 (ac AY532665) Sarafend(ac AY688948) and goshawk Austria 36110 (2009) (acHM015884) were reused from our previous works [22ndash24]The strains USUV Germany 2011 (BH65) (ac HE599647)YFV 17D JEV TBEV Langat Malaysia 1956 (ac AF253419)and Murray Valley encephalitis virus (MVEV) were reusedfrom another work [25] USUV Austria 2001 (93901) andGermany 2011 (93901) were taken from [26] and JEVNakay-ama (ac EF571853) and TBEV Neudoerfl (ac U27495) from[27]

Additionally the following strains were obtained fromthe Health Protection Agency Salisbury United KingdomWNV lineage 2 MB 1957 SLEV YFV ldquoFrench Neurotroprdquoand LolIl (780) JEV Nakamura was kindly provided byA Mullbacher (John Curtin School of Medical ResearchCanberra Australia) RNA from ZIKV and DENV 1 2 3and 4 viruses was provided by P Despres (Flavivirus UnitInstitute Pasteur Paris France) while F Hufert and M Wei-dmann (Institute for Virology Gottingen Germany) kindlyprovided the viruses TBEV Absettarov 1951 (ac AF091005)TBEV Aina 1963 (ac AF091006) and TBEV Hypr 1953 (acU39292)

RNA was extracted with the RNeasy Mini Kit or theQIAamp Viral RNA Mini Kit (QIAGEN Hilden Germany)according to the manufacturerrsquos protocol A synthetic RNAwas used as internal extraction control (IC) [28] The NY99RNAwas used to create an external calibrator curve which inturnwas calibrated using the 51015840UTRWNV-specific RT-qPCR[22]

22 Selection of Primers andProbes Nearly 200 completeFlavi-virus genome sequences were obtained from the NCBINucleotide database by the end of 2010 Very similar se-quences (more than 98 identity)were excludedThe sequen-ces were aligned using CLUSTAL X [29] (accessed fromBioEdit software v7053) [30] and VectorNTI Advanced v10(Invitrogen Carlsbad CA USA) This alignment was manu-ally curated using both the nucleotide and the deduced aminoacid sequences and extended by adding partial sequences toreach a total of more than 400 so as to represent the NS5gene of all known species includingmost subtypes or lineages(a regularly updated version is available at Flavivirus GitHubsite (httpsgithubcomqPCR4virFlavivirus))

VisualOligoDeg (httpsgithubcomqPCR4virVisualO-liDeg) was used to facilitate visualization and selection ofappropriate oligonucleotides hybridizing to sequences of agivenTheNS5 regions (spanning nucleotides 9040 to 9305 ofAF196835) of the aligned sequences were imported into thisworkbook and were manually classified into major groups(MB mosquito borne TB tick borne and insect-only) virusgroups (JEVG YFVG TBEVG etc) species (WNV JEVYFV TBEV etc) and in some cases lineages (like WNV-1 orWNV-2 etc) or genotypes

Alongside species-specific RT-PCRs [31] broader genus-specific RT-PCR protocols have been also reported [1921 32 33] A genus-specific Taq-Man RT-PCR for Fla-vivirus detection was modified [21] with primer sequen-ces optimized using VisualOligoDeg It targets conservedflanking regions with an internal region with sufficient vari-ability to enable virus identification by sequencing the ampli-conThe original sequences were adapted to consistently am-plify most of the Flavivirus members resulting in degener-ate primers PFlav-fAAR (TACAACATGATGGGAAA-GAGAGAGAARAA from 9040 to 9068 of AF196835) andPFlavrKR (GTGTCCCAKCCRGCTGTGTCATC from posi-tions 9305 to 9283 of AF196835)

A total of 50 probes with a Tm around 55∘Cwere selectedA second set of sequences was also selected as a replace-ment in case of failure of first-set sequences The candidatesequences were submitted to the manufacturer for a final insilico evaluation of properties (homogenous hybridizationdiscriminatory potential etc) As a result all 84 sequenceswere found suitable for inclusion in the production of themicroarray (Table S01 in Supplementary Material availableonline at httpsdoiorg10115520174248756)

23 Detection andQuantificationUsing RT-qPCR Aone-stepSYBR Green-based RT-qPCR with melting curve analysiswas developed The QuantiTect SYBR Green RT-PCR Kit(QIAGEN Hilden Germany) was used following the manu-facturerrsquos instructions Briefly each primer (PFlav-fAAR andPFlav-rKR) was 51015840-biotinylated during the initial synthesis(Eurofins Genomics Ebersberg Germany) and used at afinal concentration of 08 120583M in a final reaction volume of25 120583L including 5120583L of RNA sample solution Real-time RT-qPCR was carried out on a CFX96 real-time PCR detectionsystem (Bio-Rad Laboratories Hercules USA) The thermalcycling profiles are presented in Table 1 Species-specific RT-qPCR were also used to determine the sensitivity of the
















3 Results




15

25

35

MV

V

Den

gue 4

WN

V 1

a (Eg

ypt)

WN

V 1

a (D

akar

)W

NV

1a (

NY9

9)D

engu

e 1W

NV

1b

(Kun

jin)

Den

gue 2

WN

V 2

(MB

1957

)JE

V (N

akam

ura)

SLEV

WN

V 2

(Sar

afen

d)W

NV

2 (U

gand

a 37)

WN

V 3

(Hun

gary

200

4)Zi

ka V

JEV

(Nak

ayam

a)U

sutu

VTB

EV (L

anga

at)

Den

gue 3

TBEV

(Ain

a)TB

EV (H

ypr)

TBEV

(Neu

doumlrfl

)TB

EV (A

bset

taro

v)YF

V (1

7D)

YFV

(Fre

nch

Neu

rotro

p)Lo

Ill



USUV

JEV

TBEV

WNVUg

NY99

Is081

Aus08

It08

Negunspecific

Melt peak


500

1000

1500

2000

minusd

(RFU

)dt















aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33

JEjj

35

JEjj

37

JEVG

Igf

42

JEVG

Ig-a

roaj

a41

JEVG

Ntj

a53

Mea

ban

ja8

3

Mos

qFlav

f70

MV

EVJE

VGM

Vja

48

Pow

VGf

81

RioB

rVG

f75

-75

SeaB

TBja

72

SLEV

jj5

1

TBV

f56

TBV

f58

TBVG

f79

-77

TBVG

f27

7

Tyul

eniy

f83

Usu

tuV

j55

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

WNV1aNY x 10-4

WNV1aNY x 10-4

WNV1aNY x 10-1

WNV1aNY x 10-1

000

020

030

050

070

090

010

040

060

080

JEj

34

JEjj

36

JEVG

Igf

39

JEVG

Ig-a

roaj

a40

JEVG

MV

f47

JEVG

Zif

46-

45

Mos

qFlav

3f

71-7

0

MV

EVJE

VGM

Vj4

9

NoV

ertH

ostf

69

RioB

rVG

1f2

84

RioB

rVG

ja7

4

SLEV

ja5

0

SLEV

jj5

2

TBV

f57

TBVG

f76

TBVG

f80

-77

TBVG

f27

8

Usu

tuV

f54

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(a)

000

010

020

030

040

050

060

070

080

090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

WNV2Ug x 10-4

WNV2Ug x 10-4

WNV2Ug x 10-1

WNV2Ug x 10-1

(b)

000010020030040050060070080090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

MVV15 x 10-3

MVV15 x 10-3

MVV15 x 10-1

MVV15 x 10-1

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(c)

Figure 3 Continued


aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

TBLang18x10-3

TBLang18x10-3

TBLang18x10-1

TBLang18x10-1

000010020030040050060070080090

(d)



4 Discussion






054 040 027 013 000

054 040 027 013 000






MVV15 x 10-1









YFVFN



WNV2Ug x10-4 55∘C

WNVUg x10-4 50∘C

MVV10-3 55∘C

MVV x 10-3 50∘C

Usutu10-3 55∘C

Usutu x 10-3 50∘C



SLEV x 10-3 55∘C

SLEV x10-3 50∘C

YFVFN x10-3 55∘C

YFVFN X10-3 50∘C






JENmJENm




JENy2013C0901-Bchip

JENy2013C0901-Bchip







JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

A09

01-

Gch

ipW

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1W

NV

1aIs

981

AN

SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99


WN

V1

aNY9

9x1

0-4

55∘ C


(a)







WNV1aDakar


WNV1aNY-4 x229A12

Usutux275C01xH02



Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

x10

-4W

NV

1aN

Y99

10-4

x28

0E01

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

E01

WN

V1a

Dak

ar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1a

NY-

4 x

229

A12

Usu

tux

275C

01x

H02

Usu

tuIt

a12

AN

SES2

013

x280

D02

Ustu

36

Usu

tux

10-5

x27

3D01

Usu

tu-3

6x10

-2x

321D

02x

322D

02

Usu

tu3

3 x1

0-3

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a


WN

V1

aNY9

9x1

0-4

55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

mix

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

x280

G01

mix

Usu

tux

10-5

WN

V2

Ug3

7x1

0-4

x280

F01

WN

V2

Ug3

710

000c

_mlQ

CMD

2013

x279

D02

WN

V2

Ug

x10-

4W

NV

1aN

Y-4

x299

9A

12W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

V2

Austr

ia

WN

V2

Ug3

7x1

0-4

x273

A01

WN

V2

Ug

x 10

-1W

NV

2Sa

raf

Usu

tuW

NV

2Sa

rafe

nd-6

2x1

0-2

x321

E02

x322

E02

Usu

tu x

10-5

Exp

251

02-G

266

0730

1061

0_7

WN

VD

akar

WN

VM

BW

NV

MB

WN

V1a

NY9

9M

VV

-15

14x1

0-3

x321

E01

x322

E01

Mix

Usu

tu x

10-5

+WN

VN

Yx10

-4M

ix U

sutu

x10

-4W

NV

1aN

Y99

x10-

4x2

80E0

1W

NV

2Au

s08

AN

SES2

013

x280

F03

WN

V2

Aus0

8A

NSE

S201

3x2

80D

03W

NV

2Au

s08

AN

SES2

013

x280

B02

WN

V1a

NY9

9x1

0-4

x273

B01

WN

V2

Sara

f

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

VH

eja1

0+6c

_ml-Q

CMD

2013

x279

C02

WN

V1a

-NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V2

Aus0

8A

NSE

S201

3x2

80A

03

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02W

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1U

sutu

36

WN

V2

Ug3

710

0000

c01-

B26

6072

9106

10_2

Usu

tu x10

-350∘ C

WN

V2

Ug

x10

-455∘ C

WN

V1

aNY9

9x1

0-4

55∘ C

WN

V2

Ug

x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















3 Results




15

25

35

MV

V

Den

gue 4

WN

V 1

a (Eg

ypt)

WN

V 1

a (D

akar

)W

NV

1a (

NY9

9)D

engu

e 1W

NV

1b

(Kun

jin)

Den

gue 2

WN

V 2

(MB

1957

)JE

V (N

akam

ura)

SLEV

WN

V 2

(Sar

afen

d)W

NV

2 (U

gand

a 37)

WN

V 3

(Hun

gary

200

4)Zi

ka V

JEV

(Nak

ayam

a)U

sutu

VTB

EV (L

anga

at)

Den

gue 3

TBEV

(Ain

a)TB

EV (H

ypr)

TBEV

(Neu

doumlrfl

)TB

EV (A

bset

taro

v)YF

V (1

7D)

YFV

(Fre

nch

Neu

rotro

p)Lo

Ill



USUV

JEV

TBEV

WNVUg

NY99

Is081

Aus08

It08

Negunspecific

Melt peak


500

1000

1500

2000

minusd

(RFU

)dt















aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33

JEjj

35

JEjj

37

JEVG

Igf

42

JEVG

Ig-a

roaj

a41

JEVG

Ntj

a53

Mea

ban

ja8

3

Mos

qFlav

f70

MV

EVJE

VGM

Vja

48

Pow

VGf

81

RioB

rVG

f75

-75

SeaB

TBja

72

SLEV

jj5

1

TBV

f56

TBV

f58

TBVG

f79

-77

TBVG

f27

7

Tyul

eniy

f83

Usu

tuV

j55

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

WNV1aNY x 10-4

WNV1aNY x 10-4

WNV1aNY x 10-1

WNV1aNY x 10-1

000

020

030

050

070

090

010

040

060

080

JEj

34

JEjj

36

JEVG

Igf

39

JEVG

Ig-a

roaj

a40

JEVG

MV

f47

JEVG

Zif

46-

45

Mos

qFlav

3f

71-7

0

MV

EVJE

VGM

Vj4

9

NoV

ertH

ostf

69

RioB

rVG

1f2

84

RioB

rVG

ja7

4

SLEV

ja5

0

SLEV

jj5

2

TBV

f57

TBVG

f76

TBVG

f80

-77

TBVG

f27

8

Usu

tuV

f54

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(a)

000

010

020

030

040

050

060

070

080

090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

WNV2Ug x 10-4

WNV2Ug x 10-4

WNV2Ug x 10-1

WNV2Ug x 10-1

(b)

000010020030040050060070080090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

MVV15 x 10-3

MVV15 x 10-3

MVV15 x 10-1

MVV15 x 10-1

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(c)

Figure 3 Continued


aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

TBLang18x10-3

TBLang18x10-3

TBLang18x10-1

TBLang18x10-1

000010020030040050060070080090

(d)



4 Discussion






054 040 027 013 000

054 040 027 013 000






MVV15 x 10-1









YFVFN



WNV2Ug x10-4 55∘C

WNVUg x10-4 50∘C

MVV10-3 55∘C

MVV x 10-3 50∘C

Usutu10-3 55∘C

Usutu x 10-3 50∘C



SLEV x 10-3 55∘C

SLEV x10-3 50∘C

YFVFN x10-3 55∘C

YFVFN X10-3 50∘C






JENmJENm




JENy2013C0901-Bchip

JENy2013C0901-Bchip







JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

A09

01-

Gch

ipW

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1W

NV

1aIs

981

AN

SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99


WN

V1

aNY9

9x1

0-4

55∘ C


(a)







WNV1aDakar


WNV1aNY-4 x229A12

Usutux275C01xH02



Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

x10

-4W

NV

1aN

Y99

10-4

x28

0E01

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

E01

WN

V1a

Dak

ar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1a

NY-

4 x

229

A12

Usu

tux

275C

01x

H02

Usu

tuIt

a12

AN

SES2

013

x280

D02

Ustu

36

Usu

tux

10-5

x27

3D01

Usu

tu-3

6x10

-2x

321D

02x

322D

02

Usu

tu3

3 x1

0-3

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a


WN

V1

aNY9

9x1

0-4

55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

mix

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

x280

G01

mix

Usu

tux

10-5

WN

V2

Ug3

7x1

0-4

x280

F01

WN

V2

Ug3

710

000c

_mlQ

CMD

2013

x279

D02

WN

V2

Ug

x10-

4W

NV

1aN

Y-4

x299

9A

12W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

V2

Austr

ia

WN

V2

Ug3

7x1

0-4

x273

A01

WN

V2

Ug

x 10

-1W

NV

2Sa

raf

Usu

tuW

NV

2Sa

rafe

nd-6

2x1

0-2

x321

E02

x322

E02

Usu

tu x

10-5

Exp

251

02-G

266

0730

1061

0_7

WN

VD

akar

WN

VM

BW

NV

MB

WN

V1a

NY9

9M

VV

-15

14x1

0-3

x321

E01

x322

E01

Mix

Usu

tu x

10-5

+WN

VN

Yx10

-4M

ix U

sutu

x10

-4W

NV

1aN

Y99

x10-

4x2

80E0

1W

NV

2Au

s08

AN

SES2

013

x280

F03

WN

V2

Aus0

8A

NSE

S201

3x2

80D

03W

NV

2Au

s08

AN

SES2

013

x280

B02

WN

V1a

NY9

9x1

0-4

x273

B01

WN

V2

Sara

f

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

VH

eja1

0+6c

_ml-Q

CMD

2013

x279

C02

WN

V1a

-NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V2

Aus0

8A

NSE

S201

3x2

80A

03

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02W

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1U

sutu

36

WN

V2

Ug3

710

0000

c01-

B26

6072

9106

10_2

Usu

tu x10

-350∘ C

WN

V2

Ug

x10

-455∘ C

WN

V1

aNY9

9x1

0-4

55∘ C

WN

V2

Ug

x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33

JEjj

35

JEjj

37

JEVG

Igf

42

JEVG

Ig-a

roaj

a41

JEVG

Ntj

a53

Mea

ban

ja8

3

Mos

qFlav

f70

MV

EVJE

VGM

Vja

48

Pow

VGf

81

RioB

rVG

f75

-75

SeaB

TBja

72

SLEV

jj5

1

TBV

f56

TBV

f58

TBVG

f79

-77

TBVG

f27

7

Tyul

eniy

f83

Usu

tuV

j55

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

WNV1aNY x 10-4

WNV1aNY x 10-4

WNV1aNY x 10-1

WNV1aNY x 10-1

000

020

030

050

070

090

010

040

060

080

JEj

34

JEjj

36

JEVG

Igf

39

JEVG

Ig-a

roaj

a40

JEVG

MV

f47

JEVG

Zif

46-

45

Mos

qFlav

3f

71-7

0

MV

EVJE

VGM

Vj4

9

NoV

ertH

ostf

69

RioB

rVG

1f2

84

RioB

rVG

ja7

4

SLEV

ja5

0

SLEV

jj5

2

TBV

f57

TBVG

f76

TBVG

f80

-77

TBVG

f27

8

Usu

tuV

f54

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(a)

000

010

020

030

040

050

060

070

080

090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

WNV2Ug x 10-4

WNV2Ug x 10-4

WNV2Ug x 10-1

WNV2Ug x 10-1

(b)

000010020030040050060070080090

aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5

WN

V1a

f7

WN

V2

f8

WN

V2

w2

13

WN

Vf1

YFV

1ja

67

YFV

f64

YFV

f66

YFVG

f26

2-61

MVV15 x 10-3

MVV15 x 10-3

MVV15 x 10-1

MVV15 x 10-1

WN

V1a

f11

WN

V1b

f10

WN

V2

f21

2

WN

V2

w2

14

WN

Vf9

YFV

3f6

8-67

YFV

f65

YFVG

f61

YFVG

ja6

3-61

(c)

Figure 3 Continued


aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

Den

g3

f32

Den

g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

illY

FVG

j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

7f9

8f9

9fw

23

JE2

j38

JEj

33JE

j34

JEjj

35

JEjj

36

JEjj

37

JEVG

Igf

39JE

VGIg

f42

JEVG

Ig-a

roaj

a40

JEVG

Ig-a

roaj

a41

JEVG

MV

f47

JEVG

Ntj

a53

JEVG

Zif

46-

45M

eaba

nja

83

Mos

qFlav

3f

71-7

0M

osqF

lavf

70M

VEV

JEVG

MV

j49

MV

EVJE

VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

G1

f28

4Ri

oBrV

Gf

75-7

5Ri

oBrV

Gja

74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

sutu

Vf5

4U

sutu

Vj5

5W

NV

1af

11W

NV

1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

NV

2w

213

WN

V2

w2

14W

NV

f1W

NV

f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

TBLang18x10-3

TBLang18x10-3

TBLang18x10-1

TBLang18x10-1

000010020030040050060070080090

(d)



4 Discussion






054 040 027 013 000

054 040 027 013 000






MVV15 x 10-1









YFVFN



WNV2Ug x10-4 55∘C

WNVUg x10-4 50∘C

MVV10-3 55∘C

MVV x 10-3 50∘C

Usutu10-3 55∘C

Usutu x 10-3 50∘C



SLEV x 10-3 55∘C

SLEV x10-3 50∘C

YFVFN x10-3 55∘C

YFVFN X10-3 50∘C






JENmJENm




JENy2013C0901-Bchip

JENy2013C0901-Bchip







JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

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1aN

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8x3

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2x3

18E0

1W

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1aIs

981

AN

SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

0-3

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D01

x32

2D01

WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99


WN

V1

aNY9

9x1

0-4

55∘ C


(a)







WNV1aDakar


WNV1aNY-4 x229A12

Usutux275C01xH02



Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

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NV

1aN

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10-4

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0E01

WN

V1a

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3x2

80A

02

WN

V1a

NY9

9x1

0-4

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WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

E01

WN

V1a

Dak

ar

WN

V1a

Dak

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V1a

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229

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Ustu

36

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10-5

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02x

322D

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Usu

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3 x1

0-3

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a


WN

V1

aNY9

9x1

0-4

55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

mix

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tux

10-4

WN

V2

Ug3

7x1

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WN

V1a

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9x1

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mix

WN

V2

Ug3

7x1

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WN

V1a

NY9

9x1

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mix

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10-5

WN

V2

Ug3

7x1

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WN

V2

Ug3

710

000c

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CMD

2013

x279

D02

WN

V2

Ug

x10-

4W

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1aN

Y-4

x299

9A

12W

NV

Hej

a120

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pRT

stWN

V11

04

WN

V2

Austr

ia

WN

V2

Ug3

7x1

0-4

x273

A01

WN

V2

Ug

x 10

-1W

NV

2Sa

raf

Usu

tuW

NV

2Sa

rafe

nd-6

2x1

0-2

x321

E02

x322

E02

Usu

tu x

10-5

Exp

251

02-G

266

0730

1061

0_7

WN

VD

akar

WN

VM

BW

NV

MB

WN

V1a

NY9

9M

VV

-15

14x1

0-3

x321

E01

x322

E01

Mix

Usu

tu x

10-5

+WN

VN

Yx10

-4M

ix U

sutu

x10

-4W

NV

1aN

Y99

x10-

4x2

80E0

1W

NV

2Au

s08

AN

SES2

013

x280

F03

WN

V2

Aus0

8A

NSE

S201

3x2

80D

03W

NV

2Au

s08

AN

SES2

013

x280

B02

WN

V1a

NY9

9x1

0-4

x273

B01

WN

V2

Sara

f

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

VH

eja1

0+6c

_ml-Q

CMD

2013

x279

C02

WN

V1a

-NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V2

Aus0

8A

NSE

S201

3x2

80A

03

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02W

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1aN

Y99-

8x3

11E1

2x3

18E0

1U

sutu

36

WN

V2

Ug3

710

0000

c01-

B26

6072

9106

10_2

Usu

tu x10

-350∘ C

WN

V2

Ug

x10

-455∘ C

WN

V1

aNY9

9x1

0-4

55∘ C

WN

V2

Ug

x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


aroa

JEVG

Igf

44-3

9D

eng

1f3

29

Den

g1

f33

0D

eng

2w

231

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g3

f32

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g4

f31

8D

eng

4w

219

Den

gf1

7Ed

ge_H

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j59

Edge

_Hill

YFV

Gj

60f4

3-16 f8

6f8

7f8

8f9

0f9

1f9

2f9

3f9

4f9

5f9

6f9

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23

JE2

j38

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33JE

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JEjj

36

JEjj

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JEVG

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39JE

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f42

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a40

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Ig-a

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a41

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MV

f47

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Ntj

a53

JEVG

Zif

46-

45M

eaba

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83

Mos

qFlav

3f

71-7

0M

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lavf

70M

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JEVG

MV

j49

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VGM

Vja

48

NoV

ertH

ostf

69

Pow

VGf

81Ri

oBrV

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f28

4Ri

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75-7

5Ri

oBrV

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74

SeaB

TBja

72

SLEV

ja5

0SL

EVjj

51

SLEV

jj5

2TB

Vf5

6TB

Vf5

7TB

Vf5

8TB

VGf

76TB

VGf

79-7

7TB

VGf

80-7

7TB

VGf2

77

TBVG

f27

8Ty

ulen

iyf8

3U

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sutu

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5W

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1af

11W

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1af

7W

NV

1bf

10W

NV

2f8

WN

V2

f21

2W

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2w

213

WN

V2

w2

14W

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f1W

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f9YF

V1

ja6

7YF

V3

f68-

67YF

Vf6

4YF

Vf6

5YF

Vf6

6YF

VGf

61YF

VGf2

62-

61YF

VGja

63-

61

TBLang18x10-3

TBLang18x10-3

TBLang18x10-1

TBLang18x10-1

000010020030040050060070080090

(d)



4 Discussion






054 040 027 013 000

054 040 027 013 000






MVV15 x 10-1









YFVFN



WNV2Ug x10-4 55∘C

WNVUg x10-4 50∘C

MVV10-3 55∘C

MVV x 10-3 50∘C

Usutu10-3 55∘C

Usutu x 10-3 50∘C



SLEV x 10-3 55∘C

SLEV x10-3 50∘C

YFVFN x10-3 55∘C

YFVFN X10-3 50∘C






JENmJENm




JENy2013C0901-Bchip

JENy2013C0901-Bchip







JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

A09

01-

Gch

ipW

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1W

NV

1aIs

981

AN

SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99


WN

V1

aNY9

9x1

0-4

55∘ C


(a)







WNV1aDakar


WNV1aNY-4 x229A12

Usutux275C01xH02



Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

x10

-4W

NV

1aN

Y99

10-4

x28

0E01

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

E01

WN

V1a

Dak

ar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1a

NY-

4 x

229

A12

Usu

tux

275C

01x

H02

Usu

tuIt

a12

AN

SES2

013

x280

D02

Ustu

36

Usu

tux

10-5

x27

3D01

Usu

tu-3

6x10

-2x

321D

02x

322D

02

Usu

tu3

3 x1

0-3

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a


WN

V1

aNY9

9x1

0-4

55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

mix

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

x280

G01

mix

Usu

tux

10-5

WN

V2

Ug3

7x1

0-4

x280

F01

WN

V2

Ug3

710

000c

_mlQ

CMD

2013

x279

D02

WN

V2

Ug

x10-

4W

NV

1aN

Y-4

x299

9A

12W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

V2

Austr

ia

WN

V2

Ug3

7x1

0-4

x273

A01

WN

V2

Ug

x 10

-1W

NV

2Sa

raf

Usu

tuW

NV

2Sa

rafe

nd-6

2x1

0-2

x321

E02

x322

E02

Usu

tu x

10-5

Exp

251

02-G

266

0730

1061

0_7

WN

VD

akar

WN

VM

BW

NV

MB

WN

V1a

NY9

9M

VV

-15

14x1

0-3

x321

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10-5

+WN

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ix U

sutu

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1aN

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1W

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2Au

s08

AN

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013

x280

F03

WN

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8A

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S201

3x2

80D

03W

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2Au

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013

x280

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V1a

NY9

9x1

0-4

x273

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V2

Sara

f

WN

V1a

Dak

ar-9

1x1

0-3

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x32

2D01

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VH

eja1

0+6c

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CMD

2013

x279

C02

WN

V1a

-NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V2

Aus0

8A

NSE

S201

3x2

80A

03

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02W

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1U

sutu

36

WN

V2

Ug3

710

0000

c01-

B26

6072

9106

10_2

Usu

tu x10

-350∘ C

WN

V2

Ug

x10

-455∘ C

WN

V1

aNY9

9x1

0-4

55∘ C

WN

V2

Ug

x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


054 040 027 013 000

054 040 027 013 000






MVV15 x 10-1









YFVFN



WNV2Ug x10-4 55∘C

WNVUg x10-4 50∘C

MVV10-3 55∘C

MVV x 10-3 50∘C

Usutu10-3 55∘C

Usutu x 10-3 50∘C



SLEV x 10-3 55∘C

SLEV x10-3 50∘C

YFVFN x10-3 55∘C

YFVFN X10-3 50∘C






JENmJENm




JENy2013C0901-Bchip

JENy2013C0901-Bchip







JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

A09

01-

Gch

ipW

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1W

NV

1aIs

981

AN

SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

0-3

x321

D01

x32

2D01

WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99


WN

V1

aNY9

9x1

0-4

55∘ C


(a)







WNV1aDakar


WNV1aNY-4 x229A12

Usutux275C01xH02



Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

x10

-4W

NV

1aN

Y99

10-4

x28

0E01

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

E01

WN

V1a

Dak

ar

WN

V1a

Dak

ar-9

1x1

0-3

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D01

x32

2D01

WN

V1a

NY-

4 x

229

A12

Usu

tux

275C

01x

H02

Usu

tuIt

a12

AN

SES2

013

x280

D02

Ustu

36

Usu

tux

10-5

x27

3D01

Usu

tu-3

6x10

-2x

321D

02x

322D

02

Usu

tu3

3 x1

0-3

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a


WN

V1

aNY9

9x1

0-4

55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

mix

Usu

tux

10-4

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

mix

WN

V2

Ug3

7x1

0-4

WN

V1a

NY9

9x1

0-4

x280

G01

mix

Usu

tux

10-5

WN

V2

Ug3

7x1

0-4

x280

F01

WN

V2

Ug3

710

000c

_mlQ

CMD

2013

x279

D02

WN

V2

Ug

x10-

4W

NV

1aN

Y-4

x299

9A

12W

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Hej

a120

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pRT

stWN

V11

04

WN

V2

Austr

ia

WN

V2

Ug3

7x1

0-4

x273

A01

WN

V2

Ug

x 10

-1W

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2Sa

raf

Usu

tuW

NV

2Sa

rafe

nd-6

2x1

0-2

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x322

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Usu

tu x

10-5

Exp

251

02-G

266

0730

1061

0_7

WN

VD

akar

WN

VM

BW

NV

MB

WN

V1a

NY9

9M

VV

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14x1

0-3

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Mix

Usu

tu x

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VN

Yx10

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ix U

sutu

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1aN

Y99

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1W

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2Au

s08

AN

SES2

013

x280

F03

WN

V2

Aus0

8A

NSE

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3x2

80D

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2Au

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AN

SES2

013

x280

B02

WN

V1a

NY9

9x1

0-4

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B01

WN

V2

Sara

f

WN

V1a

Dak

ar-9

1x1

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D01

x32

2D01

WN

VH

eja1

0+6c

_ml-Q

CMD

2013

x279

C02

WN

V1a

-NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V2

Aus0

8A

NSE

S201

3x2

80A

03

WN

V1a

Is98

1A

NSE

S201

3x2

80A

02W

NV

1aN

Y99-

8x3

11E1

2x3

18E0

1U

sutu

36

WN

V2

Ug3

710

0000

c01-

B26

6072

9106

10_2

Usu

tu x10

-350∘ C

WN

V2

Ug

x10

-455∘ C

WN

V1

aNY9

9x1

0-4

55∘ C

WN

V2

Ug

x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



JENmJENm




JENy2013C0901-Bchip

JENy2013C0901-Bchip







JEV

Nak

ayam

ax10

-4x

273C

01JE

Ny

JEN

mJE

Nm

JEV

Nak

5x

10-3

mpl

exx

x27

9G03

x27

6C06

JEV

Nak

ayam

aAN

SES2

013

x280

G02

2013

D10

01-

Cch

ip20

13B

020

1-Fc

hip

2605

H12

03-

Hch

ip20

13G

030

1-Ac

hip

2535

B05

02-

Bch

ipJE

Ny

2013

C09

01-

Bch

ipJE

Ny

2013

C09

01-

Bch

ip

2535

A09

01-

Gch

ipW

NV

1aN

Y99-

8x3

11E1

2x3

18E0

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1aIs

981

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SES2

013

x280

A02

WN

V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

911

cop

x210

A03

Rin

g Te

st201

101

WN

V1a

NY

x10-

4x2

73B0

1W

NV

Hej

a120

00co

pRT

stWN

V11

04

WN

VD

akar

WN

V1a

Dak

ar-9

1x1

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WN

V1

NY-

4x2

29A

12W

NV

1aN

Y99


WN

V1

aNY9

9x1

0-4

55∘ C


(a)







WNV1aDakar


WNV1aNY-4 x229A12

Usutux275C01xH02



Usutu33 x10-3

Ustu36

Usutu-36x10-2x321D02x322D02

Usutux10-5x273D01

mxU

sutu

x10

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1aN

Y99

10-4

x28

0E01

WN

V1a

Is98

1A

NSE

S201

3x2

80A

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V1a

NY9

9x1

0-4

x280

A03

WN

V1a

NY9

911

cop

x210

A03

Rin

gTes

t201

101

WN

V1a

NY9

910

00c_

mlQ

CMD

2013

x27

9A02

WN

V1a

NY9

9-8

x311

E12

x318

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WN

V1a

Dak

ar

WN

V1a

Dak

ar-9

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V1a

NY-

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Usu

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36

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10-5

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Usu

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WN

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aNY9

9x1

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55∘ C

Usutu x10-3 55∘C

Usu

tu x10

-355∘ C

Dissimilarity

000 070

(b)

Figure 5 Continued







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

mix

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WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







WNV2SarafUsutu



WNVMBWNVMB

WNV1aNY99MVV-1514x10-3x321E01x322E01







Usutu36WNV2Ug37100000c01-B266072910610_2

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0-4

55∘ C

WN

V2

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x10

-450∘ C

Usutu x10-3 50∘C

WNV2Ug x10-4 55∘C


WNV2Ug x10-4 50∘C

Dissimilarity

000 070

(c)



5 Conclusion



Disclosure






Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References

























































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

a novel pan-flavivirus detection and identification assay based...

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