Non-polio enteroviruses in faeces of children diagnosed with acute flaccid paralysis in Nigeria

ABSTRACTBackgroundThe need to investigate the contribution of non-polio enteroviruses to acute flaccid paralysis (AFP) cannot be over emphasized as we move towards a poliovirus free world. Hence, we aim to identify non-polio enteroviruses recovered from the faeces of children diagnosed with AFP in Nigeria.MethodsNinety-six isolates, (95 unidentified and one previously confirmed Sabin poliovirus 3) recovered on RD cell culture from the stool of children <15 years old diagnosed with AFP in 2014 were analyzed. All isolates were subjected to RNA extraction, cDNA synthesis and three different PCR reactions (one panenterovirus 5′-UTR and two VP1 amplification assays). VP1 amplicons were then sequenced isolates identified.Results93.75% (90/96) of the isolates were detected by at least one of the three assays as an enterovirus. Precisely, 79.17% (76/96), 6.25% (6/96), 7.295% (7/96) and 6.25% (6/96) of the isolates were positive for both, positive and negative, negative and positive, as well as negative for both the 5′-UTR and VP1 assays, respectively. In this study, sixty-nine (69) of the 83 VP1 amplicons sequenced were identified as 27 different enterovirus types. The most commonly detected were CV-B3 (10 isolates) and EV-B75 (5 isolates). Specifically, one, twenty-four and two of the enterovirus types identified in this study belong to EV-A, EV-B and EV-C respectively.DiscussionThis study reports the circulating strains of 27 non-polio enterovirus types in Nigerian children with AFP in 2014 and Nigerian strains of CV-B2, CV-B4, E17, EV-B80, EV-B73, EV-B97, EV-B93, EV-C99 and EV-A120.

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Abundance of Enterovirus C in RD-L20B cell culture negative stool samples from Acute Flaccid Paralysis cases in Nigeria is geographically defined

10.1101/222935 ◽

2017 ◽

Author(s):

E Donbraye ◽

O.I. Olasunkanmi ◽

B.A. Opabode ◽

T.R. Ishola ◽

T.O.C. Faleye ◽

...

Keyword(s):

Cell Culture ◽

Phylogenetic Analysis ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Geographical Region ◽

Flaccid Paralysis ◽

Cdna Synthesis ◽

Faecal Samples ◽

Stool Samples ◽

Culture Negative

ABSTRACTWe recently showed that Enteroviruses (EVs); majorly species Cs (EV-Cs) were present in about 46.7% of faecal samples from children <15 years old diagnosed with Acute Flaccid Paralysis (AFP) in Nigeria but declared to be EV free by the RD-L20B cell culture based algorithm. In this study, we investigated whether this observed preponderance of EVs (and EV-Cs) in such samples vary by geographical region.In all, 108 samples (i.e. 54 paired stool suspensions from 54 AFP cases) previously confirmed negative for EVs by the WHO recommended RD-L20B cell culture based algorithm were analyzed in this study. The 108 samples were made into 54 pools (27 each from Northwest [NW] and Southsouth [SS] Nigeria). All samples were subjected to RNA extraction, cDNA synthesis and the WHO recommended seminestedPCR (snPCR) assay and its modifications. All amplicons were sequenced, and enteroviruses identified using the enterovirus genotyping tool and phylogenetic analysis.Altogether, EVs were detected in 16 (29.63%) of the 54 samples screened but successfully identified in 14 (25.93%): 10 from NW- and 4 from SS-Nigeria. Precisely, one (7.14%), two (14.29%) and 11 (78.57%) of the strains detected were EV-A, EV-B and EV-C respectively. The 10 strains from NW-Nigeria are 7 EV types and include CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from SS-Nigeria include E31, CV-A1, EV-C99 and EV-C116. EV-C99 is the only EV type that was detected in both NW- and SS-Nigeria.The results of this study showed that the preponderance of EVs and consequently EV-Cs in AFP samples declared to be EV free by the RD-L20B cell culture based algorithm vary by geographical region in Nigeria. It further confirmed the EV-B bias of the RD-L20B cell culture based algorithm.

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Identification of previously untypable RD cell line isolates and detection of EV-A71 genotype C1 in a child with AFP in Nigeria

10.1101/334094 ◽

2018 ◽

Cited By ~ 1

Author(s):

M.O. Adewumi ◽

T.O.C. Faleye ◽

C.O. Okeowo ◽

A.M. Oladapo ◽

J. Oyathelemhi ◽

...

Keyword(s):

Cell Culture ◽

Phylogenetic Analysis ◽

Cell Line ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Sub Saharan Africa ◽

Flaccid Paralysis ◽

Cdna Synthesis ◽

Pcr Assay ◽

Sub Saharan

AbstractWe previously attempted to identify 96 nonpolio enteroviruses (EVs) recovered on RD cell culture from children <15 years with acute flaccid paralysis (AFP) in Nigeria. We succeeded in identifying 69 of the isolates. Here, we describe an attempt to identify the remaining 27 isolates.Twenty-six (the 27thisolate was exhausted) isolates that could not be typed previously were further analyzed. All were subjected to RNA extraction, cDNA synthesis, enterovirus 5‟-UTR– VP2 PCR assay and a modified VP1 snPCR assay. Both the 5’-UTR – VP2 and VP1 amplicons were sequenced, isolates identified and subjected to phylogenetic analysis.Twenty of the 26 isolates analyzed were successfully identified. Altogether, 23 EV strains were recovered. Thesebelong to 11 EV (one EVA, nine EVB and one EVC) types which were EVA71 genotype C1 (1 strain), CVB3 (7 strains), CVB5 (1 strain), E5 (2 strain), E11 (3 strains), E13 (2 strain), E19 (1 strain), E20 (1 strain), E24 (2 strains), EVB75 (1 strain) and EVC99 (2 strains). Of the 11 EV types, the 5’-UTR-VP2 assay identified seven while the VP1 assay identified 10. Both assays simultaneously detected 7 of the 11 EV types identified in this study with 100% congruence.In this study we identified 20 of 26 samples that were previously untypable. In addition, we provided evidence that suggests that a clade of EVA71 genotype C1 might have been circulating in sub-Saharan Africa since 2008. Finally, we showed that the 5’-UTR-VP2 assay might be as valuable as the VP1 assay in EV identification.

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Isolation and Identification of Enterovirus D111 in a Child with Acute Flaccid Paralysis in Nigeria

10.1101/479873 ◽

2018 ◽

Author(s):

T.O.C. Faleye ◽

M.O. Adewumi ◽

O.T. Olayinka ◽

J.A Adeniji

Keyword(s):

Cell Culture ◽

Cell Line ◽

Cell Lines ◽

Illumina Sequencing ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Flaccid Paralysis ◽

Isolation And Identification ◽

Test Kit ◽

Group A

ABSTRACTThe WHO recommended cell-culture-based algorithm requires enterovirus (EV) isolates to produce reproducible cytopathic effect (R-CPE) in RD and/or L20b cell lines. Samples with non-reproducible CPE (NR-CPE) are considered negative for EVs. We investigated whether there could be EVs lurking in samples with NR-CPE.Fifty-nine (59) cell culture supernatants (CCS) (collected between 2016 and 2017) recovered from RD and L20b cell culture tubes with NR-CPE, were analyzed in this study. The tubes had been previously inoculated with stool suspension from children (<15 years) in Nigeria with acute flaccid paralysis (AFP). All CCS were screened for Enteric Adenoviruses and group A Rotavirus using a rapid immunochromatographic test kit. Subsequently, they were passaged in HEp-2 cell line. All isolates were subjected to RNA extraction, cDNA synthesis, three (5l-UTR, VP1 and EV Species C [EV-C]) different PCR assays and sequencing of amplicons. EVs were further subjected to Illumina sequencing.All CCS were negative for Adenoviruses and group A Rotaviruses. Four CCS produced R-CPE in HEp-2 cell line, three of which were positive for the 5l-UTR assay. Of the 3 isolates two and none were positive for the VP1 and EV-C assays, respectively. One of the two VP1 amplicons was successfully sequenced and identified as Echovirus 1(E1). Illumina sequencing of the three 5l- UTR positive isolates confirmed the E1 isolate and typed the remaining two as EV-Ds (94 and 111).We describe the first EV-D94 and 111 isolates of Nigerian origin. We also show that NR-CPE could sometimes be caused by EVs that do not produce R-CPE in RD and L20b cell lines but do so in other cell lines like HEp-2.

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Preponderance of Enterovirus Species C in RD-L20B cell culture negative stool samples from children diagnosed with Acute Flaccid Paralysis in Nigeria

10.1101/116004 ◽

2017 ◽

Author(s):

J.A. Adeniji ◽

A.O. Oragwa ◽

U.E. George ◽

U.I. Ibok ◽

T.O.C. Faleye ◽

...

Keyword(s):

Cell Culture ◽

Acute Flaccid Paralysis ◽

Direct Detection ◽

Rna Extraction ◽

Clinical Specimen ◽

Flaccid Paralysis ◽

Surveillance Program ◽

Two Samples ◽

Afp Surveillance ◽

Stool Samples

AbstractsRecently, a reverse transcriptase seminested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses from clinical specimen. In this study, we use the assay and its modification to screen acute flaccid paralysis (AFP) samples previously confirmed negative for enteroviruses by the RD-L20B algorithm.Thirty paired stool suspensions collected in 2015 as part of the national AFP surveillance program in different states of Nigeria were analyzed in this study. The samples were previously confirmed negative for enteroviruses by the polio laboratory in accordance with the WHO recommended RD-L20B cell culture based algorithm. Two samples previously confirmed to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, and the RT-snPCR assay and its modifications. All amplicons were sequenced and enteroviruses identified using the enterovirus genotyping tool.Overall, amplicons were recovered from the two controls and 50% (15/30) of samples screened. Fourteen were successfully typed of which, 7.1% (1/14), 21.4% (3/14), 64.3% (9/14) and 7.1% (1/14) were EV-A, EV-B, EV-C and a mixture of EV-B and C (EV-C99 and E25), respectively. The two controls were identified as EV-C99 and CV-A1, both EV-Cs. The PV-2 detected had VP1 ILE143. Hence, a vaccine strain.The results of this study showed that about 50% of enterovirus infections (including some Sabin PV2s) are being missed by the RD-L20B cell culture based algorithm. This highlights the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-Cs in Nigeria was also confirmed.

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Detection of enteroviruses by reverse-transcriptase polymerase chain reaction in cell culture negative stool specimens of patients with acute flaccid paralysis

Journal of Virological Methods ◽

10.1016/j.jviromet.2007.01.010 ◽

2007 ◽

Vol 142 (1-2) ◽

pp. 95-97 ◽

Cited By ~ 10

Author(s):

Zabih-Ollah Shoja ◽

Hamideh Tabatabai ◽

Mahboobeh Sarijloo ◽

Shohreh Shahmahmoodi ◽

Talat Mokhtari Azad ◽

...

Keyword(s):

Cell Culture ◽

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Acute Flaccid Paralysis ◽

Flaccid Paralysis ◽

Chain Reaction ◽

Stool Specimens ◽

Polymerase Chain ◽

Culture Negative

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Enterovirus-A76 of South-East Asian ancestry in a Captive Chimpanzee (Pan troglodytes) in Jos, Nigeria

10.1101/120501 ◽

2017 ◽

Author(s):

A.O. Oragwa ◽

U.E. George ◽

T.O.C. Faleye ◽

M.O. Adewumi ◽

J.A. Adeniji

Keyword(s):

Nonhuman Primates ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Cdna Synthesis ◽

Detection And Identification ◽

Two Samples ◽

Mona Monkey ◽

Phosphate Buffered Saline ◽

East Asian Ancestry ◽

Wild Life

ABSTRACTWe recently detected EV-A119 and EV-B111 (previously shown to co-circulate between nonhuman primates (NHPs) and humans) in Nigerian children diagnosed with acute flaccid paralysis (AFP). This study was designed to investigate and catalogue EVs present in captive NHPs in Nigeria.Twenty-seven fecal samples collected from captive NHPs in a Wild Life Park and Zoological garden at Jos, Nigeria in April 2016 were analyzed in this study. Samples were resuspended in a phosphate buffered saline (PBS)/chloroform mixture, and the clarified supernatant was subjected to RNA extraction, cDNA synthesis, a Panenterovirus 5I-UTR assay, and three different enterovirus VP1 snPCR assays. All amplicons from the snPCR assays were sequenced, and enteroviruses identified using the enterovirus genotyping tool and phylogenetic analysis.Eight (29.63%) (two each from Chimpanzees, Patas Monkey, Mona Monkey and Baboon) of the 27 samples were positive for the 5I-UTR assay. One (3.70%) of the 27 samples was positive for the enterovirus VP1 snPCR assays in addition to its positivity by 5I-UTR assay. The same sample happens to be one of the two samples from Chimpanzees that tested positive for the 5I-UTR assay, and it was subsequently identified as EV-A76 of South-East Asia ancestry.This study documents the first recorded attempt to detect and identify enteroviruses in NHPs in Nigeria. It also reports the first detection and identification of EV-A76 in Nigeria and particularly in a NHP. It is of utmost importance that the enterovirus VP1 assays be improved to enable detection of EVs that have been detected in NHPs but yet to be described in humans.

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Comparison of Algorithms for the Detection of Enteroviruses in Stool Specimens from Children Diagnosed with Acute Flaccid Paralysis

Journal of Pathogens ◽

10.1155/2017/9256056 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

J. A. Adeniji ◽

F. A. Ayeni ◽

A. Ibrahim ◽

K. A. Tijani ◽

T. O. C. Faleye ◽

...

Keyword(s):

Cell Culture ◽

Rna Extraction ◽

Cdna Synthesis ◽

Detection Algorithms ◽

Paired Samples ◽

Fecal Suspension ◽

Increased Sensitivity ◽

Stool Specimens ◽

Stool Suspension ◽

Culture Dependent

This study was designed to compare both the cell culture dependent and independent enterovirus detection algorithms recommended by the WHO and assess how either might impact our perception of the diversity of enterovirus types present in a sample. Sixteen paired samples (16 isolates from RD cell culture and their corresponding stool suspension, i.e., 32 samples) from AFP cases in Nigeria were analyzed in this study. All the samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended RT-snPCR, and its modification. Amplicons were sequenced and strains identified. Enterovirus diversity was the same between the isolates and fecal suspension for the control and five of the samples. It was, however, different for the remaining 10 (62.5%) samples. Nine (CV-B4, E6, E7, E13, E14, E19, E29, EV-B75, and EV-B77) and five (CV-A1, CV-A11, CV-A13, EV-C99, and PV2) EV-B and EV-C types, respectively, were detected. Particularly, E19 and EV-B75 were only recovered from the isolates while E14, EV-B77, CV-A11, and CV-A13 were only recovered from fecal suspension. Both the cell culture dependent and independent protocols bias our perception of the diversity of enterovirus types present in a sample. Hence, effort should be directed at harmonizing both for increased sensitivity.

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Preponderance of enterovirus C in RD-L20B-cell-culture-negative stool samples from children diagnosed with acute flaccid paralysis in Nigeria

Archives of Virology ◽

10.1007/s00705-017-3466-2 ◽

2017 ◽

Vol 162 (10) ◽

pp. 3089-3101 ◽

Cited By ~ 12

Author(s):

J. A. Adeniji ◽

A. O. Oragwa ◽

U. E. George ◽

U. I. Ibok ◽

T. O. C. Faleye ◽

...

Keyword(s):

Cell Culture ◽

Acute Flaccid Paralysis ◽

Flaccid Paralysis ◽

Stool Samples ◽

Culture Negative

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Nonpolio enteroviruses can also be recovered from non-reproducible cytopathology in L20B cell line

10.1101/158626 ◽

2017 ◽

Author(s):

J. A. Adeniji ◽

U.I. Ibok ◽

O.T. Ayinde ◽

A.O. Oragwa ◽

U.E. George ◽

...

Keyword(s):

Cell Culture ◽

Cell Line ◽

Rna Extraction ◽

Cdna Synthesis ◽

Pcr Assay ◽

Fecal Suspension ◽

Coxsackievirus A20 ◽

Culture Supernatants ◽

Seminested Pcr ◽

University Of Ibadan

ABSTRACTSamples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed ‘non-reproducible CPE’. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.Six (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate).The results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

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