Comparison of Algorithms for the Detection of

Research ArticleComparison of Algorithms for the Detection ofEnteroviruses in Stool Specimens from Children Diagnosed withAcute Flaccid Paralysis

J A Adeniji12 F A Ayeni1 A Ibrahim1 K A Tijani13

T O C Faleye14 andM O Adewumi1

1Department of Virology College of Medicine University of Ibadan Ibadan Oyo State Nigeria2WHO National Polio Laboratory University of Ibadan Ibadan Oyo State Nigeria3Faculty of Veterinary Medicine University of Abuja FCT Abuja Nigeria4Department of Microbiology Faculty of Science Ekiti State University Ado-Ekiti Ekiti State Nigeria

Correspondence should be addressed to M O Adewumi adewumi1hotmailcom

Received 23 August 2017 Accepted 10 December 2017 Published 28 December 2017

Academic Editor Nongnuch Vanittanakom

Copyright copy 2017 J A Adeniji et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study was designed to compare both the cell culture dependent and independent enterovirus detection algorithmsrecommended by the WHO and assess how either might impact our perception of the diversity of enterovirus types present ina sample Sixteen paired samples (16 isolates from RD cell culture and their corresponding stool suspension ie 32 samples) fromAFP cases in Nigeria were analyzed in this study All the samples were subjected to RNA extraction cDNA synthesis the WHOrecommended RT-snPCR and its modification Amplicons were sequenced and strains identified Enterovirus diversity was thesame between the isolates and fecal suspension for the control and five of the samples It was however different for the remaining 10(625) samplesNine (CV-B4 E6 E7 E13 E14 E19 E29 EV-B75 andEV-B77) andfive (CV-A1 CV-A11 CV-A13 EV-C99 andPV2)EV-B and EV-C types respectively were detected Particularly E19 and EV-B75were only recovered from the isolates while E14 EV-B77 CV-A11 and CV-A13 were only recovered from fecal suspension Both the cell culture dependent and independent protocolsbias our perception of the diversity of enterovirus types present in a sample Hence effort should be directed at harmonizing bothfor increased sensitivity

1 Introduction

Enteroviruses (EVs) belong to genus Enterovirus in the familyPicornaviridae and order Picornavirales There are 13 speciesin the genus and the type species of the genus is speciesC which has poliovirus as its best studied member [1] EVsare nonenveloped viruses with icosahedral capsid symmetryand a diameter of 28ndash30 nM The genome is an sim75 kbsingle-stranded polyadenylated positive-strand RNA with acovalently linked viral protein (VPg) at the 51015840 terminus Thesingle open reading frame (ORF) in the genome is flanked bytwo untranslated regions (the 51015840UTR and 31015840UTR) The largepolyprotein translated from the single ORF is processed toyield four structural proteins (VP1 VP2 VP3 and VP4) andseven nonstructural proteinsThe sequence of the VP1 region

has been correlated with EV serotype [2] and is now used foridentification of EV types

Most information on enterovirus diversity that has beenmade available in the last three decades has been courtesyof the Global Polio Eradication Initiative (GPEI) Hencemost of these EV isolates (polioviruses [PVs] and non-polio enteroviruses [NPEVs]) were recovered following theWHO recommended cell culture based enterovirus detectionalgorithm [3 4] With the goal (poliovirus eradication) ofGPEI within reach there is justifiable concern about facilityassociated escape of polioviruses into the community aftereradication [5] Hence as part of the endgame strategyeffort is ongoing to restrict poliovirus research in cell cultureglobally to few facilities (referred to as essential facilities) with

HindawiJournal of PathogensVolume 2017 Article ID 9256056 9 pageshttpsdoiorg10115520179256056

2 Journal of Pathogens

Stool suspension

cDNA

First-round PCRproduct

Second-round PCRproduct

RNA extractionand

cDNA synthesis(primers AN32 minus AN35)

First-round PCR(primers 224 + 222)

Second-round PCR




AN89 + AN88 (PE)

189 + AN88 (EVA)

187 + AN88(EVB)

188 + AN88(EVCampD)

Isolates

RNA extractionand


Culture inRD cell line

Stool suspension

Isolates


cDNA



RNA extractionand



Second-round PCR




AN89 + AN88(PE)

189 + AN88(EVA)

187 + AN88(EVB)

188 + AN88(EVCampD)

RNA extractionand


(a)

(b)

Figure 1 Schematic representation of the algorithm used in this study (a) Sixteen RD cell culture isolates and their corresponding sixteenfecal suspensions were collected from theWHONational Polio Laboratory in Ibadan Nigeria (b) RNAwas extracted from all thirty-two (32)samples (RD positive isolates and their corresponding suspension) and subsequently converted to cDNAThe cDNA was used as template inthe 1st round PCR assayThe first-round PCR assay product was used as template in four different second-round PCR assays Positive samplesfor the 2nd round PCR assays were sequenced and the result was used for enterovirus identification

the infrastructure to prevent and contain facility associatedescape of the virus [5]

To facilitate implementation of this restriction in the nearfuture there has been significant motivation to develop verysensitive cell culture independent strategies for poliovirus(and other NPEVs) surveillance [6ndash8] In line with thisa cell culture independent algorithm developed by Nix etal [6] has been included in the recommended assays forenterovirus detection and identification by theWHO [9] Werecently showed [10] that this WHO recommended cell cul-ture independent enterovirus detection algorithm [9] missesout enterovirus coinfection This facilitates underestimationof a very common condition that was instrumental to thecirculating vaccine derived poliovirus 2 (cVDPV2) outbreak[11] in Nigeria that lasted almost a decade Consequentlywe have described modification of the assay to expandits capacity thereby facilitating detection and resolution ofcoinfection [10 12]

In the light of the biases [13 14] and limitations [10 12]of both the cell culture dependent [4] and independent [9]algorithms this study was designed to assess the impact of aswitch from the former to the latter in the future Further itinvestigated how these algorithms alongside the coinfection(species) resolution assay impact our perception of the diver-sity of enterovirus types present in a sample This study findsthat both the cell culture dependent [4] and independent [9]

algorithms have their strengths andweaknesses and unavoid-ably bias our perception of the diversity of enterovirus typespresent in a sample It demonstrates the need to maximizethe benefits of all available strategies in a bid to betterdescribe the diversity of enteroviruses in any sample ofinterest Finally this study documents the first descriptionof a Nigerian strain of EV-B77

2 Methodology

21 Sample Collection Sixteen RD positive isolates and theircorresponding suspensions (making 32 samples in all ie16 pairs of isolates from cell culture and stool suspension)were analyzed in this study The samples were collected fromthe WHO National Polio Laboratory in the Department ofVirology College of Medicine University of Ibadan Nigeria(subsequently referred to as the Polio Lab) Ten of the samplescame from five cases (ie double stool samples collected atleast 24 hours apart from the same case) The remaining sixsamples were single ones from six cases One of these sixsamples was previously identified and confirmed by the PolioLab as poliovirus 2 (PV-2) All the samples analyzed in thisstudy were collected as part of the National Acute FlaccidParalysis (AFP) surveillance programme The samples werecollected from children le 15 years presenting with AFPbetween July and August 2015The algorithm followed in thisstudy is depicted in Figure 1

Journal of Pathogens 3

22 RNAExtraction and cDNA Synthesis RNAwas extractedfrom isolates and suspensions independently using JenaBioscience Total RNA extraction kit (Jena Bioscience JenaGermany) following the manufacturerrsquos instructions ForcDNA synthesis Jena Bioscience SCRIPT cDNA SynthesisKit (Jena Bioscience Jena Germany) was used accordingto manufacturerrsquos instructions From the extract 525 120583L ofviral RNA was added to 475 120583L of cDNA synthesis mix The475 120583L of cDNA synthesis mix contained 2120583L of SCRIPT RTbuffer 05120583L of dNTPmix 05 120583LDTT stock solution 05 120583Lof RNase inhibitor 025 120583L of SCRIPT reverse transcriptaseand 025 120583L each primer AN32-AN35 The mixture wasincubated at 42∘C for 10min followed by 50∘C for 60 minutesin a Veriti thermal cycler (Applied Biosystems CaliforniaUSA)

23 Polymerase Chain Reaction The 1st round PCR reaction(Figure 1) was a total of 30 120583L reaction The reaction mixcontained 6 120583L of Red Load Taq 134 120583L of RNase freewater 03120583L of primers 224 and 222 and 10 120583L of cDNAThermal cycling was done in a Veriti thermal cycler (AppliedBiosystems California USA) as follows 94∘C for 3 minutesthen 45 cycles of 94∘C for 30 seconds 42∘C for 30 secondsand 60∘C for 60 seconds with ramp of 40 from 42∘C to60∘C This was then followed by 72∘C for 7 minutes and heldat 4∘C until the reaction was terminated

Four (PE-VP1-PCR EA-VP1-PCR EB-VP1-PCR and EC-VP1-PCR [9]) different second-round PCR assays were runin this study (Figure 1) The 2nd-round PCR assay was alsoa 30 120583L reaction The PCR reaction mix contained 6 120583L ofRed Load Taq 184 120583L of RNase free water 03120583L of forwardand reverse primers and 5 120583L of the first-round PCRproductThermal cycling was done in a Veriti thermal cycler (AppliedBiosystems California USA) The cycling conditions were94∘C for 3 minutes followed by 45 cycles of 94∘C for 30seconds 42∘C for 30 seconds and extension at 60∘C for 30seconds with ramp of 40 from 42∘C to 60∘CThis was thenfollowed by 72∘C for 7 minutes and subsequently held at 4∘Cuntil the reaction was terminated The PCR products wereresolved in a 2 agarose gels stained with ethidium bromideand viewed using a UV transilluminator

24 Amplicon Sequencing The amplicons of positive PCRreactions for the four second-round PCR assays were shippedtoMacrogen Inc Seoul SouthKorea where amplicon purifi-cation and sequencingwere done Sequencingwas done usingthe respective forward and reverse primers for each of thefour assays Subsequently using the enterovirus genotypingtool [15] and the sequence data the enterovirus genotype andspecies were determined

25 Nucleotide Sequences Accession Numbers The sequencesobtained from this study have been deposited in GenBankwith accession numbers MF686545-MF686568

26 Phylogenetic Analysis The CLUSTAL W programme inMEGA 5 software [16] was used with default settings toalign sequences of the enterovirus type(s) whose Nigerianstrains were first described in this study alongside those

retrieved from GenBank Subsequently a neighbor-joiningtree was constructed using the same MEGA5 software [16]with the Kimura-2 parameter model [17] and 1000 bootstrapreplicates The accession numbers of sequences retrievedfromGenBank for this analysis are indicated in the sequencesname on the phylograms

3 Results

31 Polymerase Chain Reaction (PCR) Assay The expectedsim330 bp fragment was successfully amplified for most of theassays carried out For the PE-VP1-PCR screen of the sixteenRD isolates subjected to this screen 939 (1516) werepositivewhile 750 (1216) of the corresponding suspensionswere also positive For the EA-VP1-PCR screen 750 (1216)of the RD isolates were positive as were 625 (1016) ofthe corresponding suspensions For the EB-VP1-PCR screen875 (1416) and 688 (1116) of the RD isolates and thecorresponding suspensions were positive respectively Alsofor theEC-VP1-PCR screen 50 (816) and 375 (616) of theRD isolates and the corresponding suspension were positiverespectively (Table 1)

32 Enterovirus Genotyping Of all the sixteen RD isolatesfifteen were amplified successfully sequenced and typed forthe PE-VP1-PCR screen using the enterovirus genotypingtool Their identities are as follows E7 (3 isolates) E19 (2isolates) E29 (1 isolate) EV B75 (1 isolate) CV A1 (1 isolate)E6 (2 isolates) E13 (4 isolates) and PV2 (1 isolate) For theEA-VP1-PCR screen twelve RD isolates were successfullyamplified but three were successfully typed and their iden-tities are as follows EV-C99 (1 isolate) CV-A1 (1 isolate)and PV2 (1 isolate) For the EB-VP1-PCR screen fourteenRD isolateswere successfully amplified sequenced and typedand their identities are as follows E19 (2 isolates) E7 (3isolates) E6 (2 isolates) E13 (4 isolates) E29 (1 isolate)EV-B75 (1 isolate) and CV-B4 (1 isolate) For the EC-VP1-PCR screen nine RD isolates were amplified but two weresuccessfully typed and their identities are PV2 (1 isolates)and EV-C99 (1 isolate) (Table 2) Over all ten serotypes wereidentified for the RD isolates PCR screen comprising speciesB (70) and species C (30) (Table 3)

Of the corresponding 16 suspensions twelve (1216) wereamplified but ten (1016) were successfully sequenced andtyped for the PE-VP1-PCR screen and their identities are asfollows E7 (1 strain) E13 (3 strains) E29 (1 strain) EV B77 (1strain) CVA1 (1 strain) E6 (2 strains) and PV2 (1 strain) Forthe EA-VP1-PCR screen eight suspensions were amplifiedsequenced and typed and their identities are as follows EV-C99 (2 strains) CV-A11 (1 strain) CV-A13 (2 strains) CV-A1(1 strain) PV2 (1 strain) and E29 (1 strain) For the EB-VP1-PCR screen twelve were amplified but ten were successfullysequenced and typed and their identities are as follows E13(3 strains) E14 (1 strain) E6 (2 strains) E7 (1 strain) CV-B4(1 strain) E29 (1 strain) and EV-B77 (1 strain) For the EC-VP1-PCR screen six suspensions were successfully amplifiedsequenced and typed and their identities are EV-C99 (2strains) CV-A13 (2 strains) PV2 (1 strain) and CV-A1 (1strain) (Table 2) Overall twelve serotypes were identified for


Table1Re

sults

oftheR

T-semi-n

estedPC

Rassays

done

inthisstu

dy

Sample

Isolates

Suspensio

nSpeciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Summary

PEEV

AEV

BEV

CampD

Summary

1Ca

se1a

++

++

++++

++

+minus

+++

2Case1b

+minus

+minus

++minus

minusminus

minus

3Case2a

++

+minus

+++

++

++

++++

4Ca

se2b

++

++

++++

++

++

++++

5Ca

se3a

++

++

++++

++

+minus

+++

6Ca

se3b

++

++

++++

++

++

++++

7Ca

se4a

+minus

++

+++

+minus

+minus

++8

Case

4b+

++

minus++

+minus

minusminus

minus

9Case5a

++

+minus

+++

minusminus

minusminus

10Ca

se5b

minusminus

minusminus

minusminus

minusminus

11Ca

se6

++

+minus

+++

++

+minus

+++

12Ca

se7

++

++

++++

++

+minus

+++

13Ca

se8

+minus

+minus

+++

minus+

minus++

14Ca

se9

++

++

++++

++

++

++++

15Ca

se10

++

++

++++

++

++

++++

16Con

trol

++

minus+

+++

++

minus+

+++

Totalsum

mary

1512

149

1210

116


Table2Re

sults

ofnu

cleotides

equencingandidentifi

catio

nof

enterovirusisolatesa

ndstr

ains

recoveredin

thisstu

dy

Sample

Isolates

Suspensio

nSummaryof

serotypes

Species

Speciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Serotype

identifi

catio

nPE

EVA

EVB

EVCamp

DSerotype

identifi

catio

n1lowastlowast

Case

1aE19

NU

E19

NU

E19

E13

NU

E13

E13

E13E19

EV-Blowast

2Ca

se1b

E19

E19

E19

E19

EV-B

3Ca

se2a

E7EV

C99

E7E7E

V-C9

9NU

EVC9

9NU

EVC9

9EV

-C99

E7E

V-C9

9EV

-BE

V-Clowast

4Ca

se2b

E7NU

E7EV

C99

E7E

V-C9

9NU

EVC9

9E14

EVC9

9E14EV

-C99

E7E

14E

V-C9

9EV

-BE

V-Clowast

5Ca

se3a

E6NU

E6NU

E6E6

CVA11

E6E6

CV-A11

E6C

V-A11

EV-BE

V-Clowast

6Ca

se3b

E6NU

E6NU

E6E6

CVA13

E6CV

A13

E6C

V-A13

E6C

V-A13

EV-BE

V-Clowast

7Ca

se4a

E13

E13

NU

E13

E13

E13

E13

E13

EV-B

8Ca

se4b

E13

NU

E13

E13

E13

EV-B

9lowastlowast

Case

5aE13

NU

E13

E13

E13

EV-B

10Ca

se5b

11Ca

se6

E7NU

E7E7

E7NU

E7E7

E7EV

-B12

Case

7E2

9NU

E29

NU

E29

E29

E29

E29

E29

E29

EV-B

13Ca

se8

EVB7

5EV

B75

EV-B75

EVB7

7EV

B77

EV-B77

EV-B75E

V-B7

7EV

-Blowast

14Ca

se9

CVA1

CVA1

CVB4

CV-A1CV

-B4

CVA1

CVA1

CVB4

CVA1

CV-A1CV

-B4

CV-A1CV

-B4

EV-BE

V-Clowast

15Ca

se10

E13

NU

E13

NU

E13

E13

CVA13

E13

CVA13

E13CV

-A13

E13CV

-A13

EV-BE

V-Clowast

16lowastlowastlowast

Con

trol

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

EV-C

NUnot

usablelowast

coinfectio

n


Table 3 Enterovirus types identified in this study

Enterovirus species Isolate Suspension Total Enterovirus typesEnterovirus types Number of types () Enterovirus types Number of types ()

EV-B E6 E7 E13 E19 E29EV-B75 CV-B4 7 (70) E6 E7 E13 E14 E29

EV-B77 CV-B4 7 (583) 9 (643)

EV-C EV-C99 CV-A1PV-2lowast 3 (30)

EV-C99 CV-A1CV-A11 CV-A13

PV-2lowast5 (417) 5 (357)

Total 10 (100) 12 (100) 14 (100)E echovirus EV enterovirus CV coxsackievirus PV poliovirus and lowast control PV2 italics viruses that were peculiar to the different detection algorithms

KM4865731 EVB77 PAK MEV 2118A stool AFP Pakistan 2013



KM4865711 EVB77 PAK MEV 3861B stool AFP Pakistan 2013



JX4761922 EVB77 NIV1023591LV404 stool AFP India 2010

JX5135921 EVB77 KCD85 stool Diarrhea India 2010

SOUTH-EAST ASIA 1

EVB77 stool AFP Nigeria 2015

AY2081192 EVB77 W543-122 99 stool Meningitis France 1999

AJ4930622 EVB77 CF496 99 Kosovo

AY8433021 EVB77 10394 USA Texas 1997

JN2556621 EVB77 CAF MAM 03 048 stool AFP CAF 2003




SOUTH-EAST ASIA 2

E 21 NIG AFP 2015

100

98

96

58

83

82

99

6998

85

50

005

Figure 2 Phylogram of EV-B77 The phylogram is based on an alignment of partial VP1 sequences The newly sequenced strains arehighlighted with black circleThe strain previously recovered from Sub-Saharan Africa in 2003 is indicated with black triangleThe GenBankaccession numbers of the strains are indicated in the phylogram Bootstrap values are indicated if gt50

the suspension PCR screen comprising species B (583) andspecies C (417) (Table 3)

The enterovirus diversity was shown to be the same inthe control (SN 16) and 333 (515) of the samples analyzed(Table 2) To be precise the diversity of enteroviruses was thesame between RD cell culture isolates and fecal suspensionfor the control (SN 16) Cases 4a 5b 6 7 and 9 (Table 2)Thediversity of enteroviruses was however different between RDcell culture isolates and fecal suspension for the remaining667 (1015) of the sample pairs analyzed (Table 2)

In summary fourteen different enterovirus types wereidentified in this study To be precise nine (CV-B4 E6E7 E13 E14 E19 E29 EV-B75 and EV-B77) and five (CV-A1 CV-A11 CV-A13 EV-C99 and PV2) EV-B and EV-Ctypes respectively were detected in this study (Table 3) Itis essential to emphasize that the single PV2 detected in thisstudy was the control provided by the Polio Lab

33 Phylogeny of EV-B77 This is the first EV-B77 straindescribed in Nigeria and the second in Sub-Saharan Africatill date The topology of the phylogenetic tree suggests thatthe EV-B77 detected in this study is different from all that hasbeen described till date More importantly it is different fromthe only Sub-Saharan Africa strain described till date whichwas recovered in Central Africa Republic in 2003 (Figure 2)

4 Discussion

41 Direct Detection from Clinical Specimen versus afterCulture in RD Cell Line From this study it was observedthat more enteroviruses were detected per sample by thePE-VP1-PCR assay after the suspension had been subjectedto culture in RD cell line For example Case 1b (Table 2)isolate was identified as E19 while there was no evidenceof enterovirus presence in the corresponding suspension In


the same light the isolates of Cases 4b and 5a (Table 2)were identified as E13 while there was also no evidenceof enterovirus presence in their corresponding suspensionsConsidering enteroviruses were detected in both the isolateand stool suspensions of other samples and even the ge24-hour pair of some of the samples in questions it is unlikelythat the observation is due to the presence of nonspecificinhibitors of PCR Rather this finding suggests that in thefecal suspension the virus titre might have been too low (iebelow the detection limit of the assay) to be detected directlyHowever RD cell culture appeared to increase the virus titreto a level thatwas subsequently detectable by the PE-VP1-PCRassay This thereby validates the value and use of cell culturefor enterovirus detection and identification as it significantlyincreases virus titre and thereby enhances our capacity todetect and identify the virus types present

It is however important to note that though some EVtypes were recovered in both the fecal suspension and RDcell culture some types appear to be specifically recovered ineach detection algorithm (Table 3) The enterovirus diversitywas shown to be the same between RD cell culture isolatesand fecal suspension for the control and 515 (Cases 4a5b 6 7 and 9) of the sample pairs analyzed It was how-ever different for the remaining 1015 (667) sample pairsanalyzed Particularly fascinating is the observation that insome instances the enterovirus isolate detected by the PE-VP1-PCR assay in RD cell culture supernatant is differentfrom what was detected in the corresponding suspensionFor example in Case 9 (Table 2) the isolate was identified asEVB75while EVB77 (first detection inNigeria) was identifiedin the corresponding suspension Also in Case 1a (Table 2)where the isolate was identified as E19 E13 was detected inthe corresponding stool suspension despite the fact that itis well known [18 19] and also documented in this study(Table 2) that RD cell line is both susceptible and permissiveto E13 Hence if the most abundant genome was selectivelydetected in the above stated instances these discrepanciessuggest that in either case the most abundant genome inthe suspension was different from that in the cell culturesupernatant This therefore confirms that culture in RD cellline selectively amplifies one enterovirus genome over theother in cases of coinfection [13 14 20] even in cases whereboth enterovirus types belong to the same species (Table 2)Should this observation be a valid biological phenomenonits biological basis might further illuminate how culture ofenteroviruses in RD cell line influences our perception ofthe serotype diversity in a sample Furthermore this mightalso indicate that the dynamics of enterovirus culture in RDcell line might not be representative of what happens in theintestinal tract and consequently should not be representedas such

42 The Impact of Mixture Resolving Assays Cases of enter-ovirus coinfection were established in 533 (EV-BC = 40EV-B = 20) of the samples analyzed in this study It is how-ever worthy of note that in these coinfected samples (Table 2)the enterovirus types identified with the PE-VP1-PCR assaywere mainly EV-Bs while the EV-C coinfection was majorlydetected by the species-specific assays The only exception

was in Case 9 where in both the fecal suspension and theRD cell culture isolate the enterovirus type detected using thePE-VP1-PCR assay was CVA1 while the EVB-VP1-PCR assaydetected CVB4 (Table 2) The perceived predilection of thePE-VP1-PCR assay for EV-Bs is not because the primers usedfor the assay have a bias for EV-Bs In fact similar studiesusing the same assay directly on fecal suspensions withoutculture in RD cell line show an abundance of EV-As [21]while those where the same assay was used directly on fecalsuspensions that did not show CPE in RD cell line showed anabundance of EV-Cs [12] Hence the predominance of EV-Bsas documented by the PE-VP1-PCR assay in this study mightbe due to the fact that only samples that had yielded isolatein RD cell lines were selected and analyzed in this studyConsidering the EV-B bias [13 14 20] of RD cell line thismight not be surprising This observation however suggeststhat in cases of coinfection involving different enterovirusspecies the chances of detecting all the enterovirus presentin the sample will be more likely enhanced by the addition ofspecies-specific primers to the PCR protocols For membersof the same species however combining cell culture withdirect detection from the specimen might be the strategy ofchoice (Table 2)

43 The Value of Paired Samples The need and value ofcollecting two stool samples (paired samples) about 24 hoursapart from any AFP case are well entrenched in the GPEIenterovirus detection protocols [4 9] The results of thisstudy further emphasize the importance of this principle forenterovirus surveillance For example it was observed thatE13 and E19 were detected in Case 1a but only E19 was iden-tified in Case 1b Also while E7 and EVC99 were detectedin Case 2a E14 in addition to E7 and EVC99 were detectedin Case 2b More importantly E13 was detected in Case 5awhile no enterovirus was detected in Case 5b (Table 2) Theresults of this study therefore further demonstrate that with-out paired samples many enterovirus infections would bemissed Consequently we recommended that this principlebe implemented for enterovirus surveillance in general andnot just for AFP surveillance

44 Enterovirus Detection Algorithms and the Risk of Facil-ity Associated Escape of Poliovirus after Containment Wehave shown that both the cell culture dependent [4] andindependent [9] protocols recommended by the WHO forenterovirus detection unavoidably bias our perception of thediversity of enterovirus types present in a sample (Tables2 and 3) We have also shown the shortcomings of a Pan-Enterovirus RT-PCR detection assay which is predicated onthe false assumption that coinfections are not significantwhen enterovirus infections are being considered Thoughthe anticipated need to prevent the risk of facility associ-ated escape of polioviruses cannot be overemphasized thefindings of this study suggest that enterovirologists shouldattempt to maximize the benefits of available strategies in abid to better describe the diversity of enteroviruses in anysample of interest


On the other hand effort should be put into expandingthe species [22] or serotype [23] specific nextgen sequencingstrategies that have already been developed to accommodateother enterovirus types and species They also have to beexpanded to go beyond using isolates recovered from cellculture to direct detection from clinical specimen Suchdevelopmentmight facilitate a successful switch fromcell cul-ture dependent to independent strategies without necessarilylosing out on breadth and sensitivity

Conflicts of Interest

The authors declare that no conflicts of interest exist Inaddition no information that can be used to associate theisolates analyzed in this study to any individual is includedin this manuscript

Authorsrsquo Contributions

T O C Faleye M O Adewumi and J A Adeniji performedthe study design All authors performed sample collectionand laboratory and data analysis All authors wrote revisedread and approved the final draft of the manuscript

Acknowledgments

The authors thank the WHO National Polio Laboratoryin Ibadan Nigeria for providing the anonymous isolatesanalyzed in this studyThis studywas funded by contributionsfrom the authors

References

[1] 2017 httpwwwpicornaviridaecomenterovirusenterovirushtm

[2] M S Oberste K Maher D R Kilpatrick and M A PallanschldquoMolecular evolution of the human enteroviruses correlationof serotype with VP1 sequence and application to picornavirusclassificationrdquo Journal of Virology vol 73 no 3 pp 1941ndash19481999

[3] World Health Organisation Guidelines for EnvironmentalSurveillance of Poliovirus Circulation World Health Organisa-tion Geneva Switzerland 2003

[4] World Health Organisation Polio Laboratory Manual WorldHealth Organisation Geneva Switzerland 4th edition 2004

[5] World Health Organisation Global Action Plan to MinimizePoliovirus Facility-Associated Risk World Health OrganisationGeneva Switzerland 2014

[6] W A Nix M S Oberste and M A Pallansch ldquoSensitiveseminested PCR amplification of VP1 sequences for directidentification of all enterovirus serotypes from original clinicalspecimensrdquo Journal of Clinical Microbiology vol 44 no 8 pp2698ndash2704 2006

[7] M Arita D R Kilpatrick T Nakamura et al ldquoDevelopment ofan efficient entire-capsid-coding-region amplification methodfor direct detection of poliovirus from stool extractsrdquo Journal ofClinical Microbiology vol 53 no 1 pp 73ndash78 2015

[8] A Krasota N Loginovskih O Ivanova and G LipskayaldquoDirect identification of Enteroviruses in cerebrospinal fluid ofpatients with suspected meningitis by nested PCR amplifica-tionrdquo Viruses vol 8 no 1 2016

[9] World Health Organisation Enterovirus Surveillance Guide-lines Guidelines for Enterovirus Surveillance in Support ofthe Polio Eradication Initiative World Health OrganisationGeneva Switzerland 2015

[10] T O C Faleye M O Adewumi S A Kareem et al ldquoTheimpact of a panenterovirus Vp1 assay on our perception of theenterovirus diversity landscape of a samplerdquo Journal of HumanVirology amp Retrovirology vol 4 no 3 Article ID 00134 2016

[11] C C Burns J Shaw J Jorba et al ldquoMultiple independentemergences of type 2 vaccine-derived polioviruses during alarge outbreak in northern Nigeriardquo Journal of Virology vol 87no 9 pp 4907ndash4922 2013

[12] J A Adeniji A O Oragwa U E George U I Ibok T OFaleye and M O Adewumi ldquoPreponderance of enterovirus Cin RD-L20B-cell-culture-negative stool samples from childrendiagnosed with acute flaccid paralysis in Nigeriardquo Archives ofVirology vol 162 no 10 pp 3089ndash3101 2017

[13] J A Adeniji and T O C Faleye ldquoEnterovirus C strains cir-culating in Nigeria and their contribution to the emergence ofrecombinant circulating vaccine-derived poliovirusesrdquoArchivesof Virology vol 160 no 3 pp 675ndash683 2015

[14] S A Sadeuh-Mba M Bessaud D Massenet et al ldquoHighfrequency and diversity of species C enteroviruses in Cameroonand neighboring countriesrdquo Journal of Clinical Microbiologyvol 51 no 3 pp 759ndash770 2013

[15] A Kroneman H Vennema K Deforche et al ldquoAn automatedgenotyping tool for enteroviruses and norovirusesrdquo Journal ofClinical Virology vol 51 no 2 pp 121ndash125 2011

[16] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[17] M Kimura ldquoA simple method for estimating evolutionary ratesof base substitutions through comparative studies of nucleotidesequencesrdquo Journal ofMolecular Evolution vol 16 no 2 pp 111ndash120 1980

[18] J A Adeniji and T O C Faleye ldquoIsolation and identification ofenteroviruses from sewage and sewage contaminated water inLagos Nigeriardquo Food and Environmental Virology vol 6 no 2pp 75ndash86 2014

[19] O G Oyero F D Adu and J A Ayukekbong ldquoMolecularcharacterization of diverse species enterovirus-B types fromchildren with acute flaccid paralysis and asymptomatic childrenin Nigeriardquo Virus Research vol 189 pp 189ndash193 2014

[20] J A Adeniji and T O Faleye ldquoImpact of cell lines includedin enterovirus isolation protocol on perception of nonpolioenterovirus species C diversityrdquo Journal of Virological Methodsvol 207 pp 238ndash247 2014

[21] T O C Faleye M O Adewumi B A Coker F Y Nudamajoand J A Adeniji ldquoDirect detection and identification ofenteroviruses from faeces of healthy Nigerian children using acell-culture independent RT-seminested PCR assayrdquo Advancesin Virology vol 2016 Article ID 1412838 12 pages 2016


[22] M Bessaud S A Sadeuh-Mba M-L Joffret et al ldquoWholegenome sequencing of Enterovirus species C isolates by high-throughput sequencing development of generic primersrdquo Fron-tiers in Microbiology vol 7 article 1294 2016

[23] A M Montmayeur T F F Ng A Schmidt et al ldquoHigh-throughput next-generation sequencing of poliovirusesrdquo Jour-nal of Clinical Microbiology vol 55 no 2 pp 606ndash615 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Stool suspension

cDNA



RNA extractionand



Second-round PCR




AN89 + AN88 (PE)

189 + AN88 (EVA)

187 + AN88(EVB)

188 + AN88(EVCampD)

Isolates

RNA extractionand



Stool suspension

Isolates


cDNA



RNA extractionand



Second-round PCR




AN89 + AN88(PE)

189 + AN88(EVA)

187 + AN88(EVB)

188 + AN88(EVCampD)

RNA extractionand


(a)

(b)

Figure 1 Schematic representation of the algorithm used in this study (a) Sixteen RD cell culture isolates and their corresponding sixteenfecal suspensions were collected from theWHONational Polio Laboratory in Ibadan Nigeria (b) RNAwas extracted from all thirty-two (32)samples (RD positive isolates and their corresponding suspension) and subsequently converted to cDNAThe cDNA was used as template inthe 1st round PCR assayThe first-round PCR assay product was used as template in four different second-round PCR assays Positive samplesfor the 2nd round PCR assays were sequenced and the result was used for enterovirus identification

the infrastructure to prevent and contain facility associatedescape of the virus [5]

To facilitate implementation of this restriction in the nearfuture there has been significant motivation to develop verysensitive cell culture independent strategies for poliovirus(and other NPEVs) surveillance [6ndash8] In line with thisa cell culture independent algorithm developed by Nix etal [6] has been included in the recommended assays forenterovirus detection and identification by theWHO [9] Werecently showed [10] that this WHO recommended cell cul-ture independent enterovirus detection algorithm [9] missesout enterovirus coinfection This facilitates underestimationof a very common condition that was instrumental to thecirculating vaccine derived poliovirus 2 (cVDPV2) outbreak[11] in Nigeria that lasted almost a decade Consequentlywe have described modification of the assay to expandits capacity thereby facilitating detection and resolution ofcoinfection [10 12]

In the light of the biases [13 14] and limitations [10 12]of both the cell culture dependent [4] and independent [9]algorithms this study was designed to assess the impact of aswitch from the former to the latter in the future Further itinvestigated how these algorithms alongside the coinfection(species) resolution assay impact our perception of the diver-sity of enterovirus types present in a sample This study findsthat both the cell culture dependent [4] and independent [9]

algorithms have their strengths andweaknesses and unavoid-ably bias our perception of the diversity of enterovirus typespresent in a sample It demonstrates the need to maximizethe benefits of all available strategies in a bid to betterdescribe the diversity of enteroviruses in any sample ofinterest Finally this study documents the first descriptionof a Nigerian strain of EV-B77

2 Methodology

21 Sample Collection Sixteen RD positive isolates and theircorresponding suspensions (making 32 samples in all ie16 pairs of isolates from cell culture and stool suspension)were analyzed in this study The samples were collected fromthe WHO National Polio Laboratory in the Department ofVirology College of Medicine University of Ibadan Nigeria(subsequently referred to as the Polio Lab) Ten of the samplescame from five cases (ie double stool samples collected atleast 24 hours apart from the same case) The remaining sixsamples were single ones from six cases One of these sixsamples was previously identified and confirmed by the PolioLab as poliovirus 2 (PV-2) All the samples analyzed in thisstudy were collected as part of the National Acute FlaccidParalysis (AFP) surveillance programme The samples werecollected from children le 15 years presenting with AFPbetween July and August 2015The algorithm followed in thisstudy is depicted in Figure 1









3 Results





Table1Re

sults

oftheR

T-semi-n

estedPC

Rassays

done

inthisstu

dy

Sample

Isolates

Suspensio

nSpeciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Summary

PEEV

AEV

BEV

CampD

Summary

1Ca

se1a

++

++

++++

++

+minus

+++

2Case1b

+minus

+minus

++minus

minusminus

minus

3Case2a

++

+minus

+++

++

++

++++

4Ca

se2b

++

++

++++

++

++

++++

5Ca

se3a

++

++

++++

++

+minus

+++

6Ca

se3b

++

++

++++

++

++

++++

7Ca

se4a

+minus

++

+++

+minus

+minus

++8

Case

4b+

++

minus++

+minus

minusminus

minus

9Case5a

++

+minus

+++

minusminus

minusminus

10Ca

se5b

minusminus

minusminus

minusminus

minusminus

11Ca

se6

++

+minus

+++

++

+minus

+++

12Ca

se7

++

++

++++

++

+minus

+++

13Ca

se8

+minus

+minus

+++

minus+

minus++

14Ca

se9

++

++

++++

++

++

++++

15Ca

se10

++

++

++++

++

++

++++

16Con

trol

++

minus+

+++

++

minus+

+++

Totalsum

mary

1512

149

1210

116


Table2Re

sults

ofnu

cleotides


catio

nof


ndstr

ains

recoveredin

thisstu

dy

Sample

Isolates

Suspensio

nSummaryof

serotypes

Species

Speciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Serotype

identifi

catio

nPE

EVA

EVB

EVCamp

DSerotype

identifi

catio

n1lowastlowast

Case

1aE19

NU

E19

NU

E19

E13

NU

E13

E13

E13E19

EV-Blowast

2Ca

se1b

E19

E19

E19

E19

EV-B

3Ca

se2a

E7EV

C99

E7E7E

V-C9

9NU

EVC9

9NU

EVC9

9EV

-C99

E7E

V-C9

9EV

-BE

V-Clowast

4Ca

se2b

E7NU

E7EV

C99

E7E

V-C9

9NU

EVC9

9E14

EVC9

9E14EV

-C99

E7E

14E

V-C9

9EV

-BE

V-Clowast

5Ca

se3a

E6NU

E6NU

E6E6

CVA11

E6E6

CV-A11

E6C

V-A11

EV-BE

V-Clowast

6Ca

se3b

E6NU

E6NU

E6E6

CVA13

E6CV

A13

E6C

V-A13

E6C

V-A13

EV-BE

V-Clowast

7Ca

se4a

E13

E13

NU

E13

E13

E13

E13

E13

EV-B

8Ca

se4b

E13

NU

E13

E13

E13

EV-B

9lowastlowast

Case

5aE13

NU

E13

E13

E13

EV-B

10Ca

se5b

11Ca

se6

E7NU

E7E7

E7NU

E7E7

E7EV

-B12

Case

7E2

9NU

E29

NU

E29

E29

E29

E29

E29

E29

EV-B

13Ca

se8

EVB7

5EV

B75

EV-B75

EVB7

7EV

B77

EV-B77

EV-B75E

V-B7

7EV

-Blowast

14Ca

se9

CVA1

CVA1

CVB4

CV-A1CV

-B4

CVA1

CVA1

CVB4

CVA1

CV-A1CV

-B4

CV-A1CV

-B4

EV-BE

V-Clowast

15Ca

se10

E13

NU

E13

NU

E13

E13

CVA13

E13

CVA13

E13CV

-A13

E13CV

-A13

EV-BE

V-Clowast


Con

trol

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

EV-C

NUnot

usablelowast

coinfectio

n





EV-B77 CV-B4 7 (583) 9 (643)



PV-2lowast5 (417) 5 (357)










SOUTH-EAST ASIA 1




AY8433021 EVB77 10394 USA Texas 1997





SOUTH-EAST ASIA 2

E 21 NIG AFP 2015

100

98

96

58

83

82

99

6998

85

50

005






4 Discussion















Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results





Table1Re

sults

oftheR

T-semi-n

estedPC

Rassays

done

inthisstu

dy

Sample

Isolates

Suspensio

nSpeciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Summary

PEEV

AEV

BEV

CampD

Summary

1Ca

se1a

++

++

++++

++

+minus

+++

2Case1b

+minus

+minus

++minus

minusminus

minus

3Case2a

++

+minus

+++

++

++

++++

4Ca

se2b

++

++

++++

++

++

++++

5Ca

se3a

++

++

++++

++

+minus

+++

6Ca

se3b

++

++

++++

++

++

++++

7Ca

se4a

+minus

++

+++

+minus

+minus

++8

Case

4b+

++

minus++

+minus

minusminus

minus

9Case5a

++

+minus

+++

minusminus

minusminus

10Ca

se5b

minusminus

minusminus

minusminus

minusminus

11Ca

se6

++

+minus

+++

++

+minus

+++

12Ca

se7

++

++

++++

++

+minus

+++

13Ca

se8

+minus

+minus

+++

minus+

minus++

14Ca

se9

++

++

++++

++

++

++++

15Ca

se10

++

++

++++

++

++

++++

16Con

trol

++

minus+

+++

++

minus+

+++

Totalsum

mary

1512

149

1210

116


Table2Re

sults

ofnu

cleotides


catio

nof


ndstr

ains

recoveredin

thisstu

dy

Sample

Isolates

Suspensio

nSummaryof

serotypes

Species

Speciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Serotype

identifi

catio

nPE

EVA

EVB

EVCamp

DSerotype

identifi

catio

n1lowastlowast

Case

1aE19

NU

E19

NU

E19

E13

NU

E13

E13

E13E19

EV-Blowast

2Ca

se1b

E19

E19

E19

E19

EV-B

3Ca

se2a

E7EV

C99

E7E7E

V-C9

9NU

EVC9

9NU

EVC9

9EV

-C99

E7E

V-C9

9EV

-BE

V-Clowast

4Ca

se2b

E7NU

E7EV

C99

E7E

V-C9

9NU

EVC9

9E14

EVC9

9E14EV

-C99

E7E

14E

V-C9

9EV

-BE

V-Clowast

5Ca

se3a

E6NU

E6NU

E6E6

CVA11

E6E6

CV-A11

E6C

V-A11

EV-BE

V-Clowast

6Ca

se3b

E6NU

E6NU

E6E6

CVA13

E6CV

A13

E6C

V-A13

E6C

V-A13

EV-BE

V-Clowast

7Ca

se4a

E13

E13

NU

E13

E13

E13

E13

E13

EV-B

8Ca

se4b

E13

NU

E13

E13

E13

EV-B

9lowastlowast

Case

5aE13

NU

E13

E13

E13

EV-B

10Ca

se5b

11Ca

se6

E7NU

E7E7

E7NU

E7E7

E7EV

-B12

Case

7E2

9NU

E29

NU

E29

E29

E29

E29

E29

E29

EV-B

13Ca

se8

EVB7

5EV

B75

EV-B75

EVB7

7EV

B77

EV-B77

EV-B75E

V-B7

7EV

-Blowast

14Ca

se9

CVA1

CVA1

CVB4

CV-A1CV

-B4

CVA1

CVA1

CVB4

CVA1

CV-A1CV

-B4

CV-A1CV

-B4

EV-BE

V-Clowast

15Ca

se10

E13

NU

E13

NU

E13

E13

CVA13

E13

CVA13

E13CV

-A13

E13CV

-A13

EV-BE

V-Clowast


Con

trol

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

EV-C

NUnot

usablelowast

coinfectio

n





EV-B77 CV-B4 7 (583) 9 (643)



PV-2lowast5 (417) 5 (357)










SOUTH-EAST ASIA 1




AY8433021 EVB77 10394 USA Texas 1997





SOUTH-EAST ASIA 2

E 21 NIG AFP 2015

100

98

96

58

83

82

99

6998

85

50

005






4 Discussion















Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Re

sults

oftheR

T-semi-n

estedPC

Rassays

done

inthisstu

dy

Sample

Isolates

Suspensio

nSpeciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Summary

PEEV

AEV

BEV

CampD

Summary

1Ca

se1a

++

++

++++

++

+minus

+++

2Case1b

+minus

+minus

++minus

minusminus

minus

3Case2a

++

+minus

+++

++

++

++++

4Ca

se2b

++

++

++++

++

++

++++

5Ca

se3a

++

++

++++

++

+minus

+++

6Ca

se3b

++

++

++++

++

++

++++

7Ca

se4a

+minus

++

+++

+minus

+minus

++8

Case

4b+

++

minus++

+minus

minusminus

minus

9Case5a

++

+minus

+++

minusminus

minusminus

10Ca

se5b

minusminus

minusminus

minusminus

minusminus

11Ca

se6

++

+minus

+++

++

+minus

+++

12Ca

se7

++

++

++++

++

+minus

+++

13Ca

se8

+minus

+minus

+++

minus+

minus++

14Ca

se9

++

++

++++

++

++

++++

15Ca

se10

++

++

++++

++

++

++++

16Con

trol

++

minus+

+++

++

minus+

+++

Totalsum

mary

1512

149

1210

116


Table2Re

sults

ofnu

cleotides


catio

nof


ndstr

ains

recoveredin

thisstu

dy

Sample

Isolates

Suspensio

nSummaryof

serotypes

Species

Speciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Serotype

identifi

catio

nPE

EVA

EVB

EVCamp

DSerotype

identifi

catio

n1lowastlowast

Case

1aE19

NU

E19

NU

E19

E13

NU

E13

E13

E13E19

EV-Blowast

2Ca

se1b

E19

E19

E19

E19

EV-B

3Ca

se2a

E7EV

C99

E7E7E

V-C9

9NU

EVC9

9NU

EVC9

9EV

-C99

E7E

V-C9

9EV

-BE

V-Clowast

4Ca

se2b

E7NU

E7EV

C99

E7E

V-C9

9NU

EVC9

9E14

EVC9

9E14EV

-C99

E7E

14E

V-C9

9EV

-BE

V-Clowast

5Ca

se3a

E6NU

E6NU

E6E6

CVA11

E6E6

CV-A11

E6C

V-A11

EV-BE

V-Clowast

6Ca

se3b

E6NU

E6NU

E6E6

CVA13

E6CV

A13

E6C

V-A13

E6C

V-A13

EV-BE

V-Clowast

7Ca

se4a

E13

E13

NU

E13

E13

E13

E13

E13

EV-B

8Ca

se4b

E13

NU

E13

E13

E13

EV-B

9lowastlowast

Case

5aE13

NU

E13

E13

E13

EV-B

10Ca

se5b

11Ca

se6

E7NU

E7E7

E7NU

E7E7

E7EV

-B12

Case

7E2

9NU

E29

NU

E29

E29

E29

E29

E29

E29

EV-B

13Ca

se8

EVB7

5EV

B75

EV-B75

EVB7

7EV

B77

EV-B77

EV-B75E

V-B7

7EV

-Blowast

14Ca

se9

CVA1

CVA1

CVB4

CV-A1CV

-B4

CVA1

CVA1

CVB4

CVA1

CV-A1CV

-B4

CV-A1CV

-B4

EV-BE

V-Clowast

15Ca

se10

E13

NU

E13

NU

E13

E13

CVA13

E13

CVA13

E13CV

-A13

E13CV

-A13

EV-BE

V-Clowast


Con

trol

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

EV-C

NUnot

usablelowast

coinfectio

n





EV-B77 CV-B4 7 (583) 9 (643)



PV-2lowast5 (417) 5 (357)










SOUTH-EAST ASIA 1




AY8433021 EVB77 10394 USA Texas 1997





SOUTH-EAST ASIA 2

E 21 NIG AFP 2015

100

98

96

58

83

82

99

6998

85

50

005






4 Discussion















Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Re

sults

ofnu

cleotides


catio

nof


ndstr

ains

recoveredin

thisstu

dy

Sample

Isolates

Suspensio

nSummaryof

serotypes

Species

Speciesspecific

assays

Speciesspecific

assays

SN

Cases

PEEV

AEV

BEV

CampD

Serotype

identifi

catio

nPE

EVA

EVB

EVCamp

DSerotype

identifi

catio

n1lowastlowast

Case

1aE19

NU

E19

NU

E19

E13

NU

E13

E13

E13E19

EV-Blowast

2Ca

se1b

E19

E19

E19

E19

EV-B

3Ca

se2a

E7EV

C99

E7E7E

V-C9

9NU

EVC9

9NU

EVC9

9EV

-C99

E7E

V-C9

9EV

-BE

V-Clowast

4Ca

se2b

E7NU

E7EV

C99

E7E

V-C9

9NU

EVC9

9E14

EVC9

9E14EV

-C99

E7E

14E

V-C9

9EV

-BE

V-Clowast

5Ca

se3a

E6NU

E6NU

E6E6

CVA11

E6E6

CV-A11

E6C

V-A11

EV-BE

V-Clowast

6Ca

se3b

E6NU

E6NU

E6E6

CVA13

E6CV

A13

E6C

V-A13

E6C

V-A13

EV-BE

V-Clowast

7Ca

se4a

E13

E13

NU

E13

E13

E13

E13

E13

EV-B

8Ca

se4b

E13

NU

E13

E13

E13

EV-B

9lowastlowast

Case

5aE13

NU

E13

E13

E13

EV-B

10Ca

se5b

11Ca

se6

E7NU

E7E7

E7NU

E7E7

E7EV

-B12

Case

7E2

9NU

E29

NU

E29

E29

E29

E29

E29

E29

EV-B

13Ca

se8

EVB7

5EV

B75

EV-B75

EVB7

7EV

B77

EV-B77

EV-B75E

V-B7

7EV

-Blowast

14Ca

se9

CVA1

CVA1

CVB4

CV-A1CV

-B4

CVA1

CVA1

CVB4

CVA1

CV-A1CV

-B4

CV-A1CV

-B4

EV-BE

V-Clowast

15Ca

se10

E13

NU

E13

NU

E13

E13

CVA13

E13

CVA13

E13CV

-A13

E13CV

-A13

EV-BE

V-Clowast


Con

trol

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

PV2

EV-C

NUnot

usablelowast

coinfectio

n





EV-B77 CV-B4 7 (583) 9 (643)



PV-2lowast5 (417) 5 (357)










SOUTH-EAST ASIA 1




AY8433021 EVB77 10394 USA Texas 1997





SOUTH-EAST ASIA 2

E 21 NIG AFP 2015

100

98

96

58

83

82

99

6998

85

50

005






4 Discussion















Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















EV-B77 CV-B4 7 (583) 9 (643)



PV-2lowast5 (417) 5 (357)










SOUTH-EAST ASIA 1




AY8433021 EVB77 10394 USA Texas 1997





SOUTH-EAST ASIA 2

E 21 NIG AFP 2015

100

98

96

58

83

82

99

6998

85

50

005






4 Discussion















Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Comparison of Algorithms for the Detection of