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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: faleyetemitope@gmail.com, faleyetope@yahoo.com 18
Tel: +2348023840394 19
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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538
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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
isolates. The phylogenetic tree is based on an alignment of the partial VP1 sequences. The 559
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
isolates. The phylogenetic tree is based on an alignment of the partial VP1 sequences. The 564
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
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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
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