Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.
Enterovirus infections have been associated with an array of clinical manifestations that range from aseptic meningitis through type 1 diabetes to acute flaccid paralysis (AFP) among others [
Enteroviruses are nonenveloped viruses with a diameter of 20–30 nM. Within the virion is a positive sense, single stranded RNA genome that is approximately 7,500 nt long. The genome has one open reading frame (ORF), the polyprotein product of which is autocatalytically cleaved into structural (VP1–VP4) and nonstructural (2A–3D) proteins. The ORF is flanked on both ends by untranslated regions (UTRs) and a poly-A tail at the 3′-end.
Enteroviruses belong to the genus
Besides the fact that EVB has the highest number of serotypes, it is also the most commonly detected [
Almost all previous studies documenting enterovirus diversity in Nigeria [
Recently, Nix et al.’s [
Faecal samples were collected from sixty (male = 37, female = 23) apparently healthy children aged 1 to 10 years attending public primary schools in Ibadan, Nigeria. Samples were collected from the pupils after approval and consent were secured from the school administration and the guardian or parents of the children, respectively. Stool samples were collected from each of the children into appropriately labelled sterile collection bottles. Samples were then transported to the laboratory in the Department of Virology, College of Medicine, University College Hospital, Ibadan, Nigeria, in a cooler filled with ice packs to maintain a temperature of about 4°C. On arrival at the laboratory, the stool specimens were stored at −20°C until analysis.
About one gram of each stool specimen was diluted in 3 mL phosphate buffered saline (PBS), 1 mL chloroform, and one gram of glass beads. The mixture was then vortexed for 20 minutes and thereafter centrifuged at 3000 rpm for 20 minutes. Subsequently, 2 mL of the supernatant was aliquoted in 1 mL volumes into cryovials. One vial was stored at −20°C while the other was analysed further.
JenaBioscience RNA extraction kit (Jena Bioscience, Jena, Germany) was used for viral RNA extraction according to the manufacturer’s instructions. Script cDNA synthesis kit (Jena Bioscience, Jena, Germany) was used for cDNA synthesis according to the manufacturer’s instructions. However, instead of random hexamers, primers AN32, AN33, AN34, and AN35 [
Primers were made in 25
All amplicons were shipped to Macrogen Inc., Seoul, South Korea, for purification and sequencing of only the bands of the expected size. Primers AN88 and AN89 were used for sequencing. Afterwards, the enterovirus genotyping tool [
To align the sequences described in this study with reference sequences downloaded from the GenBank, the ClustalW program in the MEGA 5 software [
All the sequences reported in this study have been deposited in GenBank under accession numbers KT717062–KT717072.
A total of 15 (25%) of the sixty (60) stool samples screened yielded the expected band size for the enterovirus VP1 gene detection RT-snPCR screen (Table
Samples positive for the enterovirus VP1 nested RT-PCR screen and the identity of enteroviruses detected in these samples.
S. number | Sample ID | Gender | Age (years) | VP1 RT-PCR | Serotype | Species |
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1 | 5 | F | 3 | Positive | Unexploitable | |
2 | 10 | M | 5 | Positive | Unexploitable | |
3 | 11 | M | 6 | Positive | CVA1 | Species C |
4 | 15 | M | 2 | Positive | CVA8 | Species A |
5 | 16 | M | 5 | Positive | EVB80 | Species B |
6 | 20 | M | 2 | Positive | CVA8 | Species A |
7 | 36 | M | 4.5 | Positive | CVA20 | Species C |
8 | 41 | M | 1.5 | Positive | EV71 | Species A |
9 | 43 | F | 1.5 | Positive | EV71 | Species A |
10 | 44 | M | 1.5 | Positive | Unexploitable | |
11 | 45 | M | 1.5 | Positive | CVA4 | Species A |
12 | 46 | F | 3.5 | Positive | CVA3 | Species A |
13 | 48 | F | 4.5 | Positive | Phage baseplate | |
14 | 50 | M | 10 | Positive | E32 | Species B |
15 | 59 | M | 10 | Positive | EVC99 | Species C |
Of the 15 amplicons subjected to sequencing, only 12 were exploitable. The remaining 3 were unexploitable due to the presence of multiple peaks in their electropherograms. Eleven (11) of the 12 exploitable sequences were successfully typed by the enterovirus genotyping tool (EGT) as Coxsackievirus A1 (CVA1) (1 strain), CVA3 (1 strain), CVA4 (1 strain), CVA8 (2 strains), CVA20 (1 strain), echovirus 32 (E32) (1 strain), Enterovirus A71 (EVA71) (2 strains), EVB80 (1 strain), and EVC99 (1 strain) (Table
With respect to CVA1, the sequences obtained from GenBank and the one described in this study clustered into five different groups with strong bootstrap support (Figure
Phylogenetic relationship of recovered CVA1 (a), CVA3 (b), and CVA4 (c) strains. The phylogram is based on alignment of the partial VP1 sequences. The newly sequenced strains and previous strains from the region are highlighted with black triangles or diamonds and circles, respectively. The GenBank accession number of the strains is indicated in the phylogram. Bootstrap values are indicated if >50%.
The two CVA8 sequences described in this study clustered with one another, with strong bootstrap support. These CVA8 sequences did not appear to be too closely related to any of the CVA8 sequences in the phylogram (Figure
Phylogenetic relationship of recovered CVA8 (a) and CVA20 (b) strains. The phylogram is based on alignment of the partial VP1 sequences. The newly sequenced strains and previous strains from the region are highlighted with black triangles or diamonds and circles, respectively. The GenBank accession number of the strains is indicated in the phylogram. Bootstrap values are indicated if >50%.
The E32 sequence described in this study did not cluster with other E32 sequences recently described in Central African Republic [
Phylogenetic relationship of recovered E32 (a) and EV71 (b) strains. The phylogram is based on alignment of the partial VP1 sequences. The newly sequenced strains and previous strains from the region are highlighted with black triangles or diamonds and circles, respectively. The GenBank accession number of the strains is indicated in the phylogram. Bootstrap values are indicated if >50%.
The single EVB80 sequence described in this study clustered, with strong bootstrap support, with others we recently found in 2014 in Nigerian children diagnosed with AFP (unpublished data). Contrary to the situation with EVB80, the EVC99 sequence described in this study was very different from the one we recently found in 2014 in Nigerian children diagnosed with AFP (unpublished data). Though they were both found in Nigeria in 2014, they appear to be most closely related to EVC99 sequences from Cameroon [
Considering that only eleven of the samples could be unequivocally shown to contain enteroviruses, the results of this study show enterovirus detection rate of 18.3% (11/60) in apparently healthy school aged children in Ibadan, Southwestern Nigeria. This is higher than the 5.5% and 10% described in previous studies from apparently healthy school aged children in Southwestern [
The results of this study showed that species A enteroviruses were more commonly detected (54.55%) than members of the other enterovirus species (Table
It can however be argued that the picture of the enterovirus diversity landscape painted by this cell-culture independent assay may just be a reflection of the primer specificities. Consequently, the tilt in the landscape towards species A members might not be a true reflection of the diversity landscape. However, considering that, as opposed to species A, C, and D which all individually have less than 30 serotypes documented, species B has over 60 serotypes documented (
The results of this study showed the presence of nine (9) different serotypes of nonpolio enteroviruses (CVA1, CVA3, CVA4, CVA8, CVA20, E32, EVA71, EVB80, and EVC99) in apparently healthy, school aged children in Ibadan, Southwestern Nigeria, in 2014 (Table
The discovery of EV71 genotype E in Nigeria in 2004 [
The EV71 strains detected in this study belonged to genotype E (Figure
Phylogenetic relationship of recovered EVB80 (a) and EVC99 (b) strains. The phylogram is based on alignment of the partial VP1 sequences. The newly sequenced strains and previous strains from the region are highlighted with black triangles and circles, respectively. The GenBank accession number of the strains is indicated in the phylogram. Bootstrap values are indicated if >50%.
The E32 isolate described in this study appeared to be more closely related to isolates from southeast Asia than those from sub-Saharan Africa (Figure
Enterovirus genotyping tool (EGT) identification of isolates and graphic view of the region of the enterovirus genome represented by the query nucleotide sequence(s). This graphic view was generated by the enterovirus genotyping tool [
CVA20
Name | Length | Genus/species | Serotype, subgenogroup | Report | Genome |
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DQ358078.1∣CVA20_serotype_CAV2 | 7444 | Enterovirus C | CVA20 | Report |
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KF303091.1∣CVA20_NIG_2012 | 660 | Enterovirus C | CVA20 | Report |
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NIE2014_CV-A20_EV-C | 354 | Enterovirus C | CVA20 | Report |
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JX426682.1∣CVA20_T08-213 | 305 | Enterovirus C | CVA20 | Report |
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JX426681.1∣CVA20_T08-166 | 303 | Enterovirus C | CVA20 | Report |
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JX426680.1∣CVA20_T08-112 | 303 | Enterovirus C | CVA20 | Report |
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JX426679.1∣CVA20_MAR-252 | 303 | Enterovirus C | CVA20 | Report |
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JX426678.1∣CVA20_MAR-250 | 305 | Enterovirus C | CVA20 | Report |
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JX426677.1∣CVA20_MAR-249 | 305 | Enterovirus C | CVA20 | Report |
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E32
Name | Length | Genus/species | Serotype, subgenogroup | Report | Genome |
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JQ411108.1∣E32_D614_M54-2009 | 259 | Enterovirus B | E32 | Report |
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AF295475.1∣E32_50_99 | 568 | Enterovirus B | E32 | Report |
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AF295518.1∣E32_PR_10 | 568 | Enterovirus B | E32 | Report |
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HQ662326.1∣E32_Mum-829 | 337 | Enterovirus B | E32 | Report |
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HQ662323.1∣E32_Mum-837 | 337 | Enterovirus B | E32 | Report |
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JN203970.1∣E32_N-990B | 372 | Enterovirus B | E32 | Report |
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JN203969.1∣E32_N-900 | 375 | Enterovirus B | E32 | Report |
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JN203968.1∣E32_N-737 | 876 | Enterovirus B | E32 | Report |
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Nix et al.’s [
There are a significant number of sequences of this sort in GenBank and some of the sub-Saharan enterovirus sequences fall into this category alongside those from other world regions (Tables
Subsequent to the completion of this study, it was observed that, in cases of enterovirus coinfection, Nix et al.’s [
The results of this study showed the presence of CVA1, CVA3, CVA4, CVA8, CVA20, E32, EVA71, EVB80, and EVC99 in Ibadan, Southwestern Nigeria, in 2014. It thereby documents the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. It further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed. The results of this study confirm that enteroviruses can be detected directly from faecal suspension using Nix et al.’s [
No information that can be used to identify participants in this study is included in this paper.
The authors declare no conflict of interests.
(1) Study design was done by Temitope Oluwasegun Cephas Faleye, Moses Olubusuyi Adewumi, and Johnson Adekunle Adeniji. (2) Sample collection was carried out by Bamidele Atinuke Coker and Felix Yasha Nudamajo. (3) Acquisition of reagents and laboratory and data analysis were the responsibility of all authors. (4) Temitope Oluwasegun Cephas Faleye wrote the first draft of the paper. (5) All Authors revised the paper. (6) And all authors read and approved the final draft.
The authors would like to thank the study participants, their school administrators, and the guardians and/or parents for their cooperation throughout the period of the study.