title: non-polio enteroviruses in faeces of children ... · background: the need to investigate the...

1

TITLE: Non-polio enteroviruses in faeces of children diagnosed with acute flaccid 1

paralysis in Nigeria. 2

AUTHORS: 3

FALEYE, TOC1,2, ADEWUMI, MO2, JAPHET, MO1,3, DAVID, OM1, OLUYEGE, AO1, 4

ADENIJI, JA2,4, FAMUREWA, O1,5. 5

Department of Microbiology, Faculty of Science, Ekiti State University, Ado-Ekiti, Ekiti 6

State, Nigeria1, Department of Virology, College of Medicine, University of Ibadan, Ibadan, 7

Oyo State, Nigeria2, Department of Microbiology, Faculty of Science, Obafemi Awolowo 8

University, Ile-Ife, Osun State, Nigeria3, WHO, National Polio Laboratory, Department of 9

Virology, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria4. 10

Microbiology Unit, Department of Biological Sciences, Kings University, P.M.B. 555, 11

Odeomu, Osun State, Nigeria5 12

13

Correspondence should be addressed to: 14

FALEYE, Temitope Oluwasegun Cephas 15

Department of Microbiology, Faculty of Science, Ekiti State University, Ado-Ekiti, Ekiti 16

State, Nigeria. 17

Email: [email protected], [email protected] 18

Tel: +2348023840394 19

20

21

22

23

24

25

26

27

28

29

30

.CC-BY-ND 4.0 International licenseacertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was notthis version posted October 30, 2016. ; https://doi.org/10.1101/084350doi: bioRxiv preprint

https://doi.org/10.1101/084350

http://creativecommons.org/licenses/by-nd/4.0/

2

ABSTRACT 31

Background: The need to investigate the contribution of non-polio enteroviruses to acute 32

flaccid paralysis (AFP) cannot be over emphasized as we move towards a poliovirus free 33

world. Hence, we aim to identify non-polio enteroviruses recovered from the faeces of 34

children diagnosed with AFP in Nigeria. 35

Methods: Ninety-six isolates, (95 unidentified and one previously confirmed Sabin 36

poliovirus 3) recovered on RD cell culture from the stool of children <15 years old diagnosed 37

with AFP in 2014 were analyzed. All isolates were subjected to RNA extraction, cDNA 38

synthesis and three different PCR reactions (one panenterovirus 5´-UTR and two VP1 39

amplification assays). VP1 amplicons were then sequenced isolates identified. 40

Results: 93.75% (90/96) of the isolates were detected by at least one of the three assays as 41

an enterovirus. Precisely, 79.17% (76/96), 6.25% (6/96), 7.295% (7/96) and 6.25% (6/96) of 42

the isolates were positive for both, positive and negative, negative and positive, as well as 43

negative for both the 5´-UTR and VP1 assays, respectively. In this study, sixty-nine (69) of the 44

83 VP1 amplicons sequenced were identified as 27 different enterovirus types. The most commonly 45

detected were CV-B3 (10 isolates) and EV-B75 (5 isolates). Specifically, one, twenty-four and two of 46

the enterovirus types identified in this study belong to EV-A, EV-B and EV-C respectively. 47

Discussion: This study reports the circulating strains of 27 non-polio enterovirus types in 48

Nigerian children with AFP in 2014 and Nigerian strains of CV-B2, CV-B4, E17, EV-B80, 49

EV-B73, EV-B97, EV-B93, EV-C99 and EV-A120. 50

KEYWORDS: AFP, Enteroviruses, Nigeria, non-polio enteroviruses, VP1 analysis 51

52

53

54



https://doi.org/10.1101/084350


3

INTRODUCTION 55

Enteroviruses (EVs) are members of the genus Enterovirus in the family Picornaviridae, 56

order Picornavirales. There are 12 species within the genus, four (EV-A to EV-D) of which 57

(alongside the Rhinoviruses) have been repeatedly found to infect humans. The enterovirus 58

capsid is a non-enveloped icosahedron with a diameter of 20–30nM. It encloses a positive 59

sense, single stranded RNA genome of ~7,500nt. The genome has untranslated regions (5´ 60

and 3 ́UTRs) flanking a single one open reading frame (ORF), whose polyprotein product is 61

auto-catalytically cleaved into eleven proteins; four structural (VP1 – VP4) and seven non-62

structural (2A – 3D) proteins. Amplification of the 5´-UTR and/or the VP1 region can be 63

used to detect the presence of enteroviruses [1-5], and the nucleotide sequence of the VP1 64

gene is used to identify enterovirus isolates [1-6]. 65

Poliovirus, the best studied member of the genus Enterovirus and the etiologic agent of 66

poliomyelitis belongs to species C within the genus. Humans remain the only known host of 67

poliovirus, thus suggesting feasibility of its eradication. Consequently, in 1988, the World 68

Health Assembly (WHA) resolved to eradicate poliomyelitis by the year 2000 [7], and the 69

Global Polio Eradication Initiative (GPEI) was established. Courtesy of the GPEI’s activities, 70

by year 2015 indigenous poliovirus transmission has been interrupted globally except in 71

Pakistan and Afghanistan (www.polioeradication.org ). This success has been the result of 72

effective vaccination and intensive surveillance. Two poliovirus vaccine preparation (Oral 73

Polio Vaccine [OPV] and Inactivated Polio Vaccine [IPV]) are currently being used by the 74

GPEI and both are very effective [8]. However, due to the reversion of OPV, as part of the 75

‘end game’ strategy, the GPEI is tilting towards IPV as we approach the final phase of polio 76

eradication [9]. 77



https://doi.org/10.1101/084350


4

Coupled with the vaccination effort is a very effective surveillance network that looks for 78

polioviruses in both sewage-contaminated water (Environmental Surveillance [ES]) and 79

children below the age of 15 years diagnosed with AFP. The ES strategy searches for 80

enteroviruses in sewage-contaminated water due to the fact that all enterovirus infected 81

individuals shed the virus in large amounts in faeces for several weeks and in turn into 82

sewage and/or sewage contaminated water [10] (Ranta et al., 2001). Therefore, ES is very 83

sensitive and can detect enterovirus isolates from both symptomatic and asymptomatic 84

individuals. The demerit of ES based strategy is that, alone, it cannot differentiate which 85

isolates are associated with clinical manifestations and hospitalisations. On the other hand, 86

AFP surveillance finds enteroviruses associated with a clinical manifestations and consequent 87

hospitalisation. However, considering that AFP surveillance detects only the < 10% of 88

enterovirus infections with clinical manifestations, the inability o f AFP surveillance to 89

see beyond the tip of the iceberg is the strength of the ES surveillances strategy. Therefore, 90

combining both strategies better illuminates our understanding of the epidemiological and 91

evolutionary trajectory of enteroviruses, particularly with respect to pathogenicity. Hence, the 92

reason the ES-AFP surveillance strategies are combined by the Global Polio Eradication 93

initiative (GPEI) in some countries [11,12]. 94

As part of the surveillance network are over 150 laboratories globally (Global Polio 95

Laboratory Network [GPLN]) that use the RD-L20B isolation protocol [11,12] for poliovirus 96

isolation. The RD-L20B isolation protocol is predicated on two different cell lines, RD (from 97

a striated muscle cancer) (McAllister et al., 1969) and L20B (a murine transgenic L cell that 98

expresses the poliovirus receptor and is consequently permissive and susceptible to 99

polioviruses) [13, 14]. As a by-product of the poliovirus surveillance programme, the GPLN 100

recovers several non-polio enteroviruses (NPEVs) on the RD cell line. 101



https://doi.org/10.1101/084350


5

The earliest molecular epidemiology study documenting NPEV from AFP cases in Nigeria 102

was carried out using the RD-L20B protocol between 2002 and 2004 [15, 16]. Subsequent 103

studies [17-21] generating nucleotide sequence data on enterovirus diversity in Nigeria have 104

been largely ES based. The only exception is a recent study [22] on enterovirus diversity in 105

healthy Nigerian children, in which a cell-culture independent enterovirus detection strategy 106

was employed. Consequently, in this study we revisit NPEVs from AFP cases in Nigeria in 107

an attempt to revise their diversity in individuals with this clinical condition in the region. 108

109

110

111

112

113

114

115

116

117

118

119

120

121



https://doi.org/10.1101/084350


6

METHODS 122

Samples 123

Ninety-six (96) RD cell culture isolates were analysed in this study. These isolates were 124

recovered from the faeces of children below the age of 15 years that were diagnosed with 125

AFP. The stool samples from which these isolates were recovered were collected in 126

accordance with the national ethical guidelines as part of the National AFP surveillance 127

programme in Nigeria and sent to the WHO National Polio Laboratory in Ibadan, Nigeria to 128

ascertain whether poliovirus is the etiologic agent of the diagnosed AFP using the WHO 129

algorithm [12]. The isolates analyzed in this study were subsequently anonymized for further 130

studies. 131

The WHO algorithm stipulates that fecal suspension from all AFP cases be inoculated into 132

RD and L20B cell lines. Isolates that show cytopathic effect (CPE) on both cell lines are 133

considered to be polioviruses, and subsequently subjected to intratypic differentiation (ITD) 134

to differentiate between the wild type and the vaccine strain. On the other hand, isolates that 135

only show CPE in RD cell line are assumed to be non-polio enteroviruses and stored away 136

since they are not of ‘urgent’ programmatic importance to the GPEI. 137

To assemble the 96 isolates analyzed in this study, from the archives, sixteen (16) isolates 138

that showed CPE in RD cell line only, were randomly selected each month from January to 139

May 2014. In June, fifteen (15) isolates were selected, and a previously identified Sabin 140

strain poliovirus 3 was added to serve as control for the study. Besides this known isolate, 141

none of the other 95 isolates had been previously identified. 142

143

144



https://doi.org/10.1101/084350


7

RNA extraction and cDNA synthesis 145

The algorithm followed in this study is as depicted in Figure 1. Using the RNA extraction kit 146

(Jena Bioscience, Jena, Germany), RNA was extracted from the isolates according to the 147

manufacturer’s instructions. Script cDNA Synthesis Kit (Jena Bioscience, Jena, Germany) 148

was used for complementary DNA (cDNA) synthesis as instructed by the manufacturer. 149

Specifically, random hexamers were used for cDNA synthesis as previously described [19]. 150

The cDNA was then stored at -80°C and used for all polymerase chain reaction (PCR) assays. 151

POLYMERASE CHAIN REACTION (PCR) ASSAYS 152

As shown in the algorithm for this study (Figure 1), three different PCR assays were run. The 153

5´-UTR and first PanEnterovirus VP1 PCR (PE-VP1-PCR) were one-step PCR assays. The 154

second PanEnterovirus VP1 PCR was a semi-nested PCR assay (PE-VP1-snPCR) and was 155

used to amplify the partial VP1 gene in those isolates for which the PE-VP1-PCR was 156

negative. All primers were made in 100µM concentrations and all PCR assays were carried 157

out in 30µL reaction volumes. All PCR products were resolved on 2% agarose gels stained 158

with ethidium bromide and viewed using a UV transilluminator. 159

5´-UTR PCR and PanEnterovirus VP1 PCR (PE-VP1-PCR) 160

Primers panent 5´-UTR F and panent 5´-UTR R [12] were used for the 5´-UTR PCR assay 161

while primers 229 and 222 [2, 6] were used for the PE-VP1-PCR assay. Each 30μL reaction 162

contained 6μL of Red Load Taq (Jena Bioscience), 5μL of cDNA, 0.3μL of each primer and 163

18.4μL of RNase-free water. Thermal cycling was done as follows: 94°C for 3 minutes, 45 164

cycles of denaturation at 94°C for 30 seconds, annealing at 42°C for 30 seconds, and 165

extension at 60°C for 30 seconds with ramp of 40% from 42°C to 60°C. This was followed 166



https://doi.org/10.1101/084350


8

by 72°C for 7 minutes, and thereafter the sample was held at 4°C until the reaction was 167

terminated. 168

PanEnterovirus VP1 semi-Nested PCR (PE-VP1-snPCR) 169

This assay is a semi-nested PCR assay. Primers 224 and 222 [5, 6] were used for the first 170

round PCR while primers AN89 and AN88 [5, 6] were used for the second round PCR assay. 171

For the first round PCR assay, the 30μL reaction contained 6μL of Red Load Taq (Jena 172

Bioscience), 5μL of cDNA, 0.3μL of each primer and 18.4μL of RNase-free water. Thermal 173

cycling was done as follows: 94°C for 3 minutes, 45 cycles of denaturation at 94°C for 30 174

seconds, annealing at 42°C for 30 seconds, and extension at 60°C for 60 seconds with ramp 175

of 40% from 42°C to 60°C. This was followed by 72°C for 7 minutes, and thereafter the 176

sample was held at 4°C until the reaction was terminated. The conditions were the same for 177

the second round PCR assay except for following modifications: Instead of cDNA, the 178

product of the first round PCR assay was used as template for the second round assay. Also 179

the extension time for the second round assay was 30 seconds as opposed to 60 seconds for 180

the first round assay. 181

182

Amplicon sequencing and enterovirus typing 183

Only amplicon of positive PCR reactions for the two VP1 PCR assays (PE-VP1-PCR and 184

PE-VP1-snPCR) were sequenced using the respective forward and reverse primers for each 185

of the assays. Amplicons were shipped to Macrogen Inc, Seoul, South Korea, for purification 186

and sequencing. Subsequently, enterovirus genotype and species were determined using the 187

Enterovirus Genotyping Tool [23]. 188

Phylogenetic Analysis 189



https://doi.org/10.1101/084350


9

The CLUSTAL W program in MEGA 5 software [24] was used with default settings to align 190

sequences of four (the two most commonly isolated and the two for which most extensive 191

nucleotide sequence data from the region exists) of the enterovirus serotype described in this 192

study alongside those retrieved from GenBank. Subsequently, a neighbor-joining tree was 193

constructed using the same MEGA 5 software with the Kimura-2 parameter model [25] and 194

1,000 bootstrap replicates. The accession numbers of sequences retrieved from GenBank for 195

this analysis are indicated in the sequence names on the phylograms. 196

197

Nucleotide sequence accession numbers 198

All sequences reported in this study have been deposited in GenBank and assigned accession 199

numbers KX580638 – KX580702 and KX656918 – KX656912. 200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215



https://doi.org/10.1101/084350


10

RESULTS 216

5´-UTR assay 217

Precisely, 86.46% (83/96) of the isolates analyzed, including the previously identified Sabin 218

3, had the expected ~114bp band size and were consequently positive for the 5´-UTR PCR 219

screen (Table 1). 220

VP1 assays (PE-VP1-PC and PE-VP1-snPCR) 221

Fifty-nine (59) of the samples analyzed in this study, excluding the previously identified 222

Sabin 3, had the ~350bp expected band size and were consequently positive for the PE-VP1-223

PCR screen. The remaining thirty-seven (37) samples were negative for the PE-VP1-PCR 224

screen. 225

Of the remaining thirty-seven (37) samples, twenty-four (24), including the previously 226

identified Sabin 3, had the ~350bp expected band size and were thereby positive for the PE-227

VP1-snPCR screen. The remaining 13 samples were negative for the PE-VP1-snPCR screen. 228

Altogether, 86.46% (83/96) of the isolates, including the previously identified Sabin 3, had 229

the expected ~350bp band size and were consequently positive for the VP1 assays. The 230

remaining 13.54% (13/96) were negative for the VP1 assays (Table 1). 231

5´-UTR versus VP1 assays 232

Overall, 93.75% (90/96) of the isolates were detected by at least one of the three assays as an 233

enterovirus. Precisely, 79.17% (76/96) of the isolates were positive for both the 5´-UTR and 234

VP1 assays. Furthermore, 6.25% (6/96) of the isolates were positive for the 5´-UTR assay 235

alone, 7.30% (7/96) were positive for only VP1 assays and 6.25% (6/96) of the isolates were 236

negative for both the 5´-UTR and VP1 assays (Table 1) 237



https://doi.org/10.1101/084350


11

Enterovirus Typing 238

Sixty-nine (69) of the 83 amplicons generated from the VP1 PCR assays and successfully 239

sequenced were identified. The remaining 14 could not be typed due to the presence of 240

multiple peaks in their electropherograms. Twenty-seven different enterovirus types were 241

identified from the 69 exploitable amplicons (Table 1). Specifically, one (1), twenty-four (24) 242

and two (2) of the enterovirus types identified in this study belong to EV-A, EV-B and EV-C 243

respectively (Table 1). 244

245

Phylogenetic Analysis 246

Of the different enterovirus types identified in this study, only four (Coxsackievirus B3 [CV-247

B3] and EV-B75 [the two most commonly isolated] and Echovirus 7 [E7] and E19 [the two 248

for which most extensive nucleotide sequence data from the region exists]) were subjected to 249

phylogenetic analysis. The CV-B3 tree (Figure 2), showed that the lineage of the CV-B3 250

genotype detected in 2002 (in green) is absolutely different from the lineage detected in 2014. 251

In addition, it is important to note that the CV-B3 sequences detected in this study appear to 252

be closely related to those from South-East Asia (Figure 2) 253

For the EV-B75, two different but closely related genotypes were detected. Both were 254

however related to EV-B75 isolates detected in Finland in 2004. One of the EV-B75 lineages 255

found in this current study shares a common ancestor with an isolate recovered from sewage 256

contaminated water collected in Northern Nigeria in 2012 (Figure 3). 257

For the E19, two different genotypes were detected. Both were also very different from the 258

E19 strains isolated in 2003 (Cluster 1). The clusters to which the isolates are most closely 259

related are indicated as 2 and 3. The single isolate from this study in cluster 2 shares a 260



https://doi.org/10.1101/084350


12

common ancestor with a lineage that was repeatedly isolated from sewage contaminated 261

water in Lagos, Southwestern Nigeria since 2010. The remaining three strains however, 262

belong to cluster 2 and are more closely related to an ES isolate recovered in Kano State, 263

Northern Nigeria in 2012 (Figure 4). 264

265

As regards, E7, two different genotypes were also detected. The clusters to which the isolates 266

are most closely related are indicated as 1 and 2. Two of the isolates from this study in 267

cluster 1 share a common ancestor with a lineage that was first isolated from sewage 268

contaminated water in Lagos, Southwestern Nigeria in 2010. The remaining two strains 269

however, belong to cluster 2 and are more closely related to isolates repeatedly recovered in 270

AFP cases in India since 2008 (Figure 5). 271

272

273

. 274

275

276

277

278

279

280

281

282

283

284



https://doi.org/10.1101/084350


13

DISCUSSION 285

In this study, we document nucleic acid sequence data for twenty-seven (27) different 286

enterovirus types circulating and particularly present in children below the age of 15 years 287

diagnosed with AFP in Nigeria in 2014. It is important to mention that prior this study, no 288

nucleic acid sequence data existed in Genbank for nine (9) of these enterovirus types from 289

Nigeria. To be precise, to the best of the authors’ knowledge, nucleic acid sequence data for 290

Nigerian strains of E17, CV-B2, CV-B4, EV-B97, EV-B80, EV-B73, EV-B93, EV-C99 and 291

EV-A120 are being reported for the first time. It is however, our opinion, that the fact that 292

these molecular sequence data are being reported for the first time does not imply new 293

introduction of these types into the region. Rather, we believe that these types had probably 294

been around for a long time. Hence not detecting them might reflect the lack of interest in 295

NPEVs because the global effort focused on eradicating polioviruses. However, as the goal of 296

poliovirus eradication nears [9], there might be an upsurge of interest in NPEVs in a bid to 297

better understand enterovirus biology and association with varying clinical conditions. 298

In this study, 95.65% (66/69) and 88.9% (24/27) of the isolates and enterovirus types 299

successfully identified, respectively, belonged to EV-B (Table 2). This is consistent with the 300

findings of other studies from the region [15, 16, 26] predicated on the RD-L20B algorithm, 301

irrespective of whether healthy children [26] or those diagnosed with AFP were investigated 302

[15, 16]. However, it was recently shown that when cell-culture bias is bypassed by the direct 303

detection of enteroviruses from stool specimen, EV-As appear to be the most preponderant in 304

the stool of healthy children from the region [22]. Furthermore, recent studies [19, 20] 305

showed that most CV-A13s (EV-Cs) circulating in the region selectively replicate, and can be 306

isolated on MCF-7, but not in RD cell line. Also, when the same sample was simultaneously 307

inoculated into RD and MCF-7 cell lines, EV-Bs and EV-Cs respectively are specifically 308



https://doi.org/10.1101/084350


14

recovered on the two cell lines [20]. In fact, we recently observed (unpublished) that when 309

AFP samples that are negative for enteroviruses using the RD-L20B enterovirus detection 310

algorithm [11] are subjected to the recently recommended [6] direct detection RT-snPCR 311

algorithm described by Nix et al., [5], EV-Cs form the majority of enteroviruses detected. In 312

addition, we also recently observed (unpublished) that about 50% of stool suspension from 313

children with AFP in the region that yield EV-Bs in RD cell line also have an EV-C member 314

present in the faecal suspension that was not detectable by the cell line. Put together, the 315

preponderance of EV-B depicted by the results of this study and previous RD-L20B based 316

studies from the region and globally should not be interpreted as an unbiased picture of the 317

diversity landscape of enteroviruses in the AFP samples that yielded the isolates analyzed. 318

Rather, it should be correctly viewed as the landscape as seen through the bias of RD cell 319

culture. 320

Though in this study we describe enteroviruses present in the stool of children diagnosed with 321

AFP, the findings of this study show the significance of merging ES and AFP data. 322

Phylogenetic analysis (Figures 3, 4 and 5) suggests that representatives of some of the 323

lineages of EV-B75, E19 and E7 detected in the AFP cases had been previously detected 324

through environmental surveillance. Interestingly, all the sequences from Nigeria in figures 4 325

and 5 except those from this study and the 2002-2004 sequences were from environmental 326

surveillance. The ES data rightly show that more lineages are circulating in the population 327

than depicted by the AFP data reported in this study. This thereby emphasizes the power of 328

ES to illuminate our understanding of the diversity of enterovirus types and lineages 329

circulating in the population. This will, consequently, further enable us to better understand 330

the evolutionary trajectory of these enterovirus types and detect their silent circulation 331

especially in the absence of clinical manifestations. 332



https://doi.org/10.1101/084350


15

Surveillance of enterovirus diversity among healthy children can also increase the power of 333

the ES-AFP surveillance strategy. For instance, it was previously shown [22] that other 334

enterovirus types not detected in this study (e.g. EV-A71 and several CV-A types) were also 335

present and circulating in Nigeria in the same year, 2014. In addition, we had shown [22] that 336

the EV-B80 detected in a healthy Nigerian child belonged to the same lineage as those 337

detected in children diagnosed with AFP in this study. We further showed [22] that the EV-338

C99 lineage found in a healthy Nigerian child is different from that described in this study. 339

Thus, though not suggested or included in the GPEI surveillance algorithm, enterovirus 340

surveillance in healthy children is useful and can significantly complement the ES-AFP 341

strategy of the GPEI. 342

The baseline nucleotide sequence data for CV-B3 and E19 circulating in the region were 343

generated in 2002 and 2003 respectively [15, 16]. Hence, the data presented in this study is a 344

re-sampling of circulating strains of these types after over a 10-year period. For both types, 345

the strains circulating when the baseline data was generated appear to have been completely 346

replaced (Figures 2 and 4). On the other hand, E7 and EV-B75 for which baseline sequence 347

data from the region was generated in 2010 [17] and 2012 [21] respectively, members of the 348

baseline lineage were still detected, however, alongside new lineages. 349

In some instances, it appears the variation in the population is seeded from another 350

population. For example, as shown for CV-B3 and E7 (Figures 2 and 5), it appears that in 351

both instances, strains from South-East Asia were imported into Nigeria and subsequently 352

detected in the faeces of children diagnosed with AFP. These suspected importations 353

corroborate what is known about poliovirus global circulation [8, 27]. What is not clear is 354

why most non-polio enterovirus lineages detected in sub-Saharan Africa are yet to be 355



https://doi.org/10.1101/084350


16

detected and described in other world regions. This observation forms the basis of the 356

regional confinement hypothesis [28]. 357

Six (6/96) of the isolates analyzed in this study were positive for the 5´-UTR screen but 358

negative for the VP1 screen. These isolates might actually be enteroviruses that have VP1 359

primer binding sites that are too divergent to be bound by the primers. Another seven (7/96) 360

isolates on the other hand were negative for the 5´-UTR screen but positive for the VP1 361

assays. Should the 5´-UTR result be the basis for selecting isolates subjected to the VP1 362

screens, all these isolates would have been missed. It is currently not clear why these isolates 363

were negative for the 5´-UTR screen. However, it is important to mention that recently, a new 364

enterovirus 5´-UTR region was described that has a large deletion spanning the region 365

amplified in this assay [29-32]. Though described in EV-C isolates, it is not clear whether 366

such constructs are present in members of other enterovirus species and whether such 367

constructs would be functional when transferred to other species through recombination in 368

the 5´-UTR region. Whatever be the case, the 5´-UTR primers used in this study could not 369

detect these isolates. Consequently, the results of this study suggests that coupling the 5´-370

UTR and VP1 assays might provide added sensitivity, required to find some divergent types. 371

Particularly, it suggests that being positive for the 5´-UTR assay should not be the basis for 372

subjecting isolates to the VP1 assays. 373

In this study, there were different groups of ‘untypable’ isolates (Table 1). The first group 374

were those positive for the 5´-UTR screen but negative for the VP1 screen and have been 375

addressed above. The second group were those positive for the VP1 assay but for which the 376

eletropherogram could not be exploited due to multiple peaks. These are likely to come from 377

cases where the children in question were co-infected with more than one type of enterovirus. 378

This is not unusual and we have more recently observed (unpublished) that in about 50% of 379



https://doi.org/10.1101/084350


17

cases, stool samples from children with AFP in Nigeria contain more than one enterovirus 380

type and/or species. 381

The third group were those negative for both the 5´-UTR and the VP1 assays. Considering we 382

detected isolates that were positive for the 5´-UTR screen but negative for the VP1 screen 383

and vice-versa, it is not difficult to conceptualize the possibility that enterovirus isolates 384

might exist that are negative for both assays. However, currently, this is only a conjecture. 385

Since RD cell line can also support replication of other enteric viruses like the adenoviruses 386

[33], these third group of ‘untypables’ might not necessarily be enteroviruses but could be 387

other viruses for which the RD cell line is both susceptible and permissive. 388

In conclusion, we identified 27 different enterovirus types present in Nigerian children with AFP 389

in 2014. To the best of our knowledge, we also document the first molecular detection and 390

identification of Nigerian strains of E17, CV-B2, CV-B4, EV-B97, EV-B80, EV-B73, EV-391

B93, EV-C99 and EV-A120. The results of this study suggest that coupling the 5´-UTR and 392

VP1 assays might provide the added sensitivity, required to detect more enterovirus types. 393

Particularly, that being positive for the 5´-UTR assay should not be the basis for subjecting 394

isolates to the VP1 assays. The results of this study also suggest that there might be 395

importation into Nigeria of enterovirus clades from other world regions, and especially 396

South-East Asia. Finally, the results of this study show that surveillance of the environment, 397

AFP and healthy children all contribute significantly towards obtaining a complete picture of 398

the diversity of enterovirus types present and circulating in any population. 399

CONFLICT OF INTERESTS 400

The authors declare that no conflict of interests exist. In addition, no information that can be 401

used to associate the isolates analyzed in this study to any individual is included in this 402

manuscript. The samples analyzed in this study were collected as part of previous 403



https://doi.org/10.1101/084350


18

independent studies and were anonymized before use in this study. Thus, this article does not 404

contain any studies with human participants performed by any of the authors. 405

ACKNOWLEDGEMENTS 406

We thank the WHO National Polio Laboratory in Ibadan, Nigeria for providing the 407

anonymous isolates analyzed in this study. This study was funded by a Tertiary Education 408

Trust Fund, Research Project Intervention, Abuja, Nigeria to FO. The funding body had no 409

role in study design; in the collection, analysis and interpretation of data; in the writing of the 410

report; and in the decision to submit the article for publication. Hence, the opinions expressed 411

in this article are solely those of the authors. 412

AUTHOR CONTRIBUTIONS 413

1. Study Design (All authors) 414

415

2. Sample Collection and laboratory analysis (FTOC, AMO, AJA) 416

417

3. Data analysis (All Authors) 418

419

4. Wrote the first draft of the Manuscript (FTOC) 420

421

5. Revised the Manuscript (All Authors) 422

423

6. Read and Approved the Final Draft (All Authors) 424

425

7. AJA and FO both supervised this study 426

427

428

429

430

431

432

433

434

435

436



https://doi.org/10.1101/084350


19

REFERENCES 437

1. Oberste, M. S., Maher, K., Kilpatrick, D. R., Pallansch, M. A. Molecular evolution 438

of the human enteroviruses: correlation of serotype with VP1 sequence and 439

application to picornavirus classification. Journal of Virology. 1999; 73(3): 1941-440

1948. 441

2. Oberste, M.S., Nix, W.A., Maher, K., Pallansch, M.A. Improved molecular 442

identification of enteroviruses by RT–PCR and amplicon sequencing. Journal of 443

Clinical Virology. 2003; 26(3): 375-377. 444

3. Oberste, M.S., Maher, K., Williams, A.J., Dybdahl-Sissoko, N., Brown, B.A., 445

Gookin, M.S., Penaranda, S., Mishrik, N., Uddin, M. and Pallansch, M.A. Species-446

specific RT-PCR amplification of human enteroviruses: a tool for rapid species 447

identification of uncharacterized enteroviruses. Journal of General Virology. 2006; 448

87: 119–128 449

4. Oberste, M.S., and Pallansch, M.A. Enteroviruses molecular detection and typing. 450

Reviews in Medical Microbiology. 2005;16: 163-171. 451

5. Nix, W.A., Oberste, M.S., Pallansch, M.A. Sensitive, Seminested PCR Amplification 452

of VP1 Sequences for Direct Identification of All Enterovirus Serotypes from Original 453

Clinical Specimens. Journal of Clinical Microbiology. 2006; 44(8): 2698–2704 454

6. World Health Organisation. Enterovirus surveillance guidelines: Guidelines for 455

enterovirus surveillance in support of the Polio Eradication Initiative, Geneva. 2015. 456

7. WHO 1988. Global eradication of poliomyelitis by the year 2000 (World Health 457

Assembly resolutionWHA41–28). 458

http://www.polioeradication.org/content/publications/19880513_resolution.pdf 459

8. Nathanson, N. and Kew O.M. From Emergence to Eradication: The Epidemiology of 460

Poliomyelitis Deconstructed. American Journal of Epidemiology. 2010; 172(11): 461

1213 – 1229. 462

9. Minor, P.D. The polio-eradication programme and issues of the end game. Journal of 463

General Virology. 2012; 93: 457–474. 464

10. Ranta, J., Hovi, T. and Arjas, E. Poliovirus Surveillance by Examining Sewage Water 465

Specimens: Studies on Detection Probability Using Simulation Models. Risk 466

Analysis. 2001; 21(6): 1087-1096. 467

11. World Health Organisation. Guidelines for environmental surveillance of poliovirus 468

circulation. Geneva. 2003. 469

12. World Health Organisation. Polio laboratory Manual. 4th Edition, Geneva. 2004. 470

13. Pipkin, P.A., Wood, D.J., Racaniello, V.R., Minor, P.D. Characterization of L cells 471

expressing the human poliovirus receptor for the specific detection of polioviruses in 472

vitro. Journal of Virological Methods. 1993; 41: 333-340 473

14. Yoshii, K., Yoneyama, T., Shimizu, H., Yoshida, H., Hagiwara, A. Sensitivity of cells 474

to poliovirus. Japanese Journal of Infectious Disease, 1999; 52: 169 475

15. Oyero, O.G. and Adu, F. D. Non-polio enteroviruses serotypes circulating in Nigeria. 476

African Journal of Medical Sciences. 2010; 39(S): 201-208. 477

16. Oyero, O. G., F. D. Adu, J. A. Ayukekbong. Molecular characterization of diverse 478

species enterovirus-B types from children with acute flaccid paralysis and 479

asymptomatic children in Nigeria. Virus Research. 2014; 189: 189-193 480

17. Adeniji, J .A. and Faleye, T.O.C. Isolation and identification of enteroviruses from 481

sewage and sewage contaminated water in Lagos, Nigeria. Food and Environmental 482

Virology. 2014a.; 6: 75-86 483



https://doi.org/10.1101/084350


20

18. Adeniji, J.A. and Faleye, T.O.C. Impact of Cell Lines Included in Enterovirus 484

Isolation Protocol on Perception of Nonpolio Enterovirus Species C Diversity. Journal 485

of Virological Methods. 2014b; 207: 238-247 486

19. Adeniji, J .A. and Faleye, T.O.C. Enterovirus C strains circulating in Nigeria and 487

their contribution to the emergence of recombinant circulating vaccine-derived 488

polioviruses. Archives of Virology. 2015; 160 (3): 675-683. 489

20. Faleye, T.O.C. and Adeniji, J.A. Nonpolio Enterovirus-C (NPEV-C) strains 490

circulating in South-Western Nigeria and their contribution to the emergence of 491

recombinant cVDPV2 lineages. British Journal of Virology. 2015a; 2(5): 68-73 492

21. Faleye T.O.C. and Adeniji J.A. Enterovirus Species B Bias of RD Cell Line and Its 493

Influence on Enterovirus Diversity Landscape. Food and Environmental Virology. 494

2015b; 7(4): 390-402. 495

22. Faleye, T.O.C., Adewumi, M.O., Coker, B.A., Nudamajo, F.Y., Adeniji, J.A. Direct 496

detection and identification of enteroviruses from faeces of healthy Nigerian children 497

using a cell-culture independent RT- semi-nested PCR assay. Advances in 498

Virology.,2016: doi:10.1155/2016/1412838. 499

23. Kroneman, A., Vennema, H., Deforche, K., Avoort, H.v.d., Penarandac, S., Oberste 500

M.S., Vinjéc, J., Koopmans, M. An automated genotyping tool for enteroviruses and 501

noroviruses. Journal of Clinical Virology. 2011; 51: 121–125 502

24. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. MEGA5: 503

molecular evolutionary genetics analysis using maximum likelihood, evolutionary 504

distance, and maximum parsimony methods. Molecular Biology and Evolution. 505

2011; 28(10): 2731-2739. 506

25. Kimura, M. A simple method for estimating evolutionary rate of base substitutions 507

through comparative studies of nucleotide sequences. Journal of Molecular 508

Evolution. 1980; 16(2): 111-120 509

26. Baba, M.M., Oderinde, B.S., Patrick, P.Z., Jarmai, M.M. Sabin and wild polioviruses 510

from apparently healthy primary school children in northeastern Nigeria. Journal of 511

Medical Virology. 2012; 84(2): 358-364. 512

27. Burns, C.C., Diop, O.M., Sutter, R.W., Kew, O.M. Vaccine Derived Polioviruses. 513

Journal of Infectious Disease. 2014; 210 (suppl 1) 514

28. Sadeuh-Mba, S. A., Bessaud, M., Massenet, D., Joffret, M. L., Endegue, M. C., 515

Njouom, R., Reynes, J. M., Rousset, D., Delpeyroux, F. High frequency and diversity 516

of species C enteroviruses in Cameroon and neighboring countries. Journal of Clinical 517

Microbiology. 2013; 51(3): 759-770. 518

29. Tapparel C, Junier T, Gerlach D, Van-Belle S, Turin L, Cordey S, et al. New 519

respiratory enterovirus and recombinant rhinoviruses among circulating 520

picornaviruses. Emerging Infectious Disease. 2009; 15: 719–26. 521

30. Yozwiak NL, Skewes-Cox P, Gordon A, Saborio S, Kuan G, Balmaseda A, et al. 522

Human enterovirus 109: a novel interspecies recombinant enterovirus isolated from a 523

case of acute pediatric respiratory illness in Nicaragua. Journal of Virology. 2010; 84: 524

9047–58. 525

31. Lukashev A.N., Drexler J.F., Kotova V.O., Amjaga E.N., Reznik V.I., Gmyl A.P., 526

Grard, G., Taty, T., Trotsenko, O.E., Leroy, E.M., Drosten C. Novel Serotypes 105 527

and 116 are members of distinct subgroups of human enterovirus C. Journal of 528

General Virology. 2012; 93: 2357–62. 529

32. Richter, J., Tryfonos, C., Panagiotou, C., Nikolaou, E.,Maria Koliou, M., 530

Christodoulou C. Newly emerging C group enteroviruses may elude diagnosis due to 531



https://doi.org/10.1101/084350


21

a Divergent 5´-UTR. International Journal of Infectious Disease. 2013; 17: e1245–532

e1248 533

33. Enomoto, M., Fujimoto,T.T., Konagaya, M., Hanaoka, N., Chikahira, M., Taniguchi, 534

K., Okabe, N. Cultivation for 21 days should be considered to isolate respiratory 535

adenoviruses from samples containing small numbers of adenoviral genomes. 536

Japanese Journal of Infectious Disease. 2010; 63(5): 338-341 537

538

539

540



https://doi.org/10.1101/084350


22

541

Figures and Tables 542

Figure 1: The algorithm used in this study. A depicts the 5´-UTR assay. B and C show the 543

two VP1 assays. While B has only one stage of PCR, C has two consecutive stages (snPCR) 544

of PCR. 545

546

Figure 2: Phylogram of genetic relationship between VP1 nucleotide sequences of CV-B3 547

isolates. The phylogenetic tree is based on an alignment of the partial VP1 sequences. The 548

CV-B3 sequences recovered in Nigeria in 2002 are indicated in green. The newly sequenced 549

strains described in this study are highlighted in red. The purple clades represent CV-B3 550

strains for South-East Asia. The blue and black clades represent CV-B3 strains from Eurasia 551

Figure 3: Phylogram of genetic relationship between VP1 nucleotide sequences of EV-552

B75 isolates. The phylogenetic tree is based on an alignment of the partial VP1 sequences. 553

The newly sequenced strains are indicated with black diamond while the 2012 strain from 554

Nigeria is indicated with a black triangle. The GenBank accession numbers and strain of the 555

isolates are indicated in the tree. Bootstrap values are indicated if >50%. SEA represents 556

South-East Asia. 557

Figure 4: Phylogram of genetic relationship between VP1 nucleotide sequences of E19 558


newly sequenced strains are indicated with black diamond. The GenBank accession numbers 560

and strain of the isolates are indicated in the tree. Bootstrap values are indicated if >50%. 561

SEA represents South-East Asia. The numbered vertical bars are for ease of reference alone. 562

Figure 5: Phylogram of genetic relationship between VP1 nucleotide sequences of E7 563


newly sequenced strains are indicated with black diamond. The GenBank accession numbers 565

and strain of the isolates are indicated in the tree. Bootstrap values are indicated if >50%. The 566

numbered vertical bars are for ease of reference alone. 567

568

569

570

571

572

573

574

575

576

577

578

579



https://doi.org/10.1101/084350


23

TABLE 1: Summary of isolates characterized in this study. Twenty-seven different enterovirus types were identified in this study. Specifically, one, 580

twenty-four and two of the enterovirus types identified in this study belong to EV-A (EV-A120), EV-B and EV-C (EV-C99, SPV-3) respectively. Where 581

indicated, the asterisk (*) links the indicated amplicon to its enterovirus type, and ‘()’ denotes the number of amplicons that were successfully 582

identified. 583

S/N MONTH ISOLATES SCREENED

POSITIVE FOR 5´-UTR SCREEN

POSITIVE FOR VP1 SCREEN

POSITIVE FOR BOTH 5´-UTR AND VP1 SCREEN

POSITIVE FOR 5´-UTR AND NEGATIVE FOR VP1 SCREEN

NEGATIVE FOR 5´-UTR AND POSITIVE FOR VP1 SCREEN

NEGATIVE FOR BOTH 5´-UTR AND VP1 SCREEN

TOTAL ISOLATES IDENTIFIED

SEROTYPES (Number of Isolates)

1 January 16 15 14 13 (12) 1 1 (0) 0 12 CV-B3 (2), E7 (3), E13 (2), E19 (1), E20 (1), E33 (1), E6 (2)

2 February 16 14 15 13 (12) 1 2 (1)* 0 13 CV-B2 (1), CV-B3 (2), CV-B4 (1), E3 (2), E12 (1), E17 (1), E20 (3)*, E21 (1), EV-C99 (1)

3 March 16 14 13 13 (11) 1 0 (0) 2 11 CV-B3 (1), CV-B4 (1), CV-B5 (2), E11 (2), E12 (1), E13 (1), E30 (1), EV-B75 (1), EV-B80 (1)

4 April 16 13 14 12 (11) 1 2 (1)* 1 12 CV-B3 (3)*, E1 (2), E11 (1), E19 (1), E21 (1), EV-B73 (1), EV-B80 (2), EV-A120 (1)

5 May 16 13 12 11 (10) 2 1 (0) 2 10 CV-B3 (2), E6 (2), E13 (1), E14 (1), E19 (1), E26 (1), EV-B75 (2)

6 June 16 14 15 14 (11) 0 1 (0) 1 11 CV-B4 (1), CV-B5 (2), E7 (1), E11 (1), E19 (1), EV-B75 (2), EV-B93 (1), EV-B97, SPV3 (1)

TOTAL 96 83 83 76 (67) 6 7 (2) 6 69 584

585

.C

C-B

Y-N

D 4.0 International license

acertified by peer review

) is the author/funder, who has granted bioR

xiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (w

hich was not

this version posted October 30, 2016.

; https://doi.org/10.1101/084350

doi: bioR

xiv preprint

https://doi.org/10.1101/084350




https://doi.org/10.1101/084350


title: non-polio enteroviruses in faeces of children ... · background: the need to investigate the...

Documents