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

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.

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

2018 ◽  
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.

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

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.

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

2017 ◽  
Vol 162 (10) ◽  
pp. 3089-3101 ◽  
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

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

2016 ◽  
Author(s):  
TOC Faleye ◽  
MO Adewumi ◽  
MO Japhet ◽  
OM David ◽  
AO Oluyege ◽  
...  
Keyword(s):  
Cell Culture ◽  
Acute Flaccid Paralysis ◽  
Rna Extraction ◽  
Flaccid Paralysis ◽  
Free World ◽  
Cdna Synthesis ◽  
Nigerian Children

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.

scholarly journals Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria is geographically defined

2018 ◽  
Vol 67 (6) ◽  
pp. 854-865 ◽  
Author(s):  
Emmanuel Donbraye ◽  
Oluwatayo Israel Olasunkanmi ◽  
Babatunde Ayoola Opabode ◽  
Temitayo Rachael Ishola ◽  
Temitope Oluwasegun Cephas Faleye ◽  
...  
Keyword(s):  
Cell Culture ◽  
Acute Flaccid Paralysis ◽  
Flaccid Paralysis ◽  
Stool Samples ◽  
Culture Negative

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

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.

scholarly journals Culture-Independent Detection of Poliovirus in Stool Samples by Direct RNA Extraction

2021 ◽  
Author(s):  
Chelsea Harrington ◽  
Hong Sun ◽  
Stacey Jeffries-Miles ◽  
Nancy Gerloff ◽  
Mark Mandelbaum ◽  
...  
Keyword(s):  
Cell Culture ◽  
World Health Organization ◽  
Surveillance System ◽  
Virus Isolation ◽  
Rna Extraction ◽  
World Health ◽  
Culture Independent ◽  
Laboratory Surveillance ◽  
Stool Samples ◽  
Health Organization

The GPLN is a global surveillance system composed of 146 laboratories in 92 countries, in each of the six World Health Organization regions. Laboratory surveillance for PV relies on virus isolation by cell culture to identify PV in stool samples from AFP cases.

Polioviruses and other enteroviruses isolated from faecal samples of patients with acute flaccid paralysis in Australia, 1996-2004

2006 ◽  
Vol 42 (6) ◽  
pp. 370-376 ◽  
Author(s):  
Heath Kelly ◽  
Kerri A Brussen ◽  
Andrew Lawrence ◽  
Elizabeth Elliot ◽  
John Pearn ◽  
...  
Keyword(s):  
Acute Flaccid Paralysis ◽  
Flaccid Paralysis ◽  
Faecal Samples

scholarly journals Metagenomic Analyses of Viruses in Stool Samples from Children with Acute Flaccid Paralysis

2009 ◽  
Vol 83 (9) ◽  
pp. 4642-4651 ◽  
Author(s):  
Joseph G. Victoria ◽  
Amit Kapoor ◽  
Linlin Li ◽  
Olga Blinkova ◽  
Beth Slikas ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Acute Flaccid Paralysis ◽  
Sequence Similarity ◽  
Pcr Amplification ◽  
Plant Viruses ◽  
Flaccid Paralysis ◽  
Human Enterovirus ◽  
Aichi Virus ◽  
Demographic Groups ◽  
Stool Samples

ABSTRACT We analyzed viral nucleic acids in stool samples collected from 35 South Asian children with nonpolio acute flaccid paralysis (AFP). Sequence-independent reverse transcription and PCR amplification of capsid-protected, nuclease-resistant viral nucleic acids were followed by DNA sequencing and sequence similarity searches. Limited Sanger sequencing (35 to 240 subclones per sample) identified an average of 1.4 distinct eukaryotic viruses per sample, while pyrosequencing yielded 2.6 viruses per sample. In addition to bacteriophage and plant viruses, we detected known enteric viruses, including rotavirus, adenovirus, picobirnavirus, and human enterovirus species A (HEV-A) to HEV-C, as well as numerous other members of the Picornaviridae family, including parechovirus, Aichi virus, rhinovirus, and human cardiovirus. The viruses with the most divergent sequences relative to those of previously reported viruses included members of a novel Picornaviridae genus and four new viral species (members of the Dicistroviridae, Nodaviridae, and Circoviridae families and the Bocavirus genus). Samples from six healthy contacts of AFP patients were similarly analyzed and also contained numerous viruses, particularly HEV-C, including a potentially novel Enterovirus genotype. Determining the prevalences and pathogenicities of the novel genotypes, species, genera, and potential new viral families identified in this study in different demographic groups will require further studies with different demographic and patient groups, now facilitated by knowledge of these viral genomes.

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