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 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 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

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.

The Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine Treatment in Resistant 2D and 3D Model Triple Negative Breast Cancer Cell Line: ABCG2 Expression Data

2021 ◽  
Vol 21 ◽  
Author(s):  
Fatma Kubra Ata ◽  
Serap Yalcin
Keyword(s):  
Breast Cancer ◽  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profile ◽  
Three Dimensional ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Xtt Assay ◽  
Gene And Protein Expression

Background: Chemotherapeutics have been commonly used in cancer treatment. Objective: In this study, the effects of Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine have been evaluated on two-dimensional (2D) (sensitive and resistance) cell lines and three dimensional (3D) spheroid structure of MDA-MB-231. The 2D cell culture lacks a natural tissue-like structural so, using 3D cell culture has an important role in the development of effective drug testing models. Furthermore, we analyzed the ATP Binding Cassette Subfamily G Member 2 (ABCG2) gene and protein expression profile in this study. We aimed to establish a 3D breast cancer model that can mimic the in vivo 3D breast cancer microenvironment. Methods: The 3D spheroid structures were multiplied (globally) using the three-dimensional hanging drop method. The cultures of the parental cell line MDA-MB-231 served as the controls. After adding the drugs in different amounts we observed a clear and well-differentiated spheroid formation for 24 h. The viability and proliferation capacity of 2D (sensitive and resistant) cell lines and 3D spheroid cell treatment were assessed by the XTT assay. Results: Cisplatin, Irinotecan, 5-Fu, and Gemcitabine-resistant MDA-MB-231 cells were observed to begin to disintegrate in a three-dimensional clustered structure at 24 hours. Additionally, RT-PCR and protein assay showed overexpression of ABCG2 when compared to the parental cell line. Moreover, MDA-MB-231 cells grown in 3D showed decreased sensitivity to chemotherapeutics treatment. Conclusion: More resistance to chemotherapeutics and altered gene expression profile was shown in 3D cell cultures when compared with the 2D cells. These results might play an important role to evaluate the efficacy of anticancer drugs, explore mechanisms of MDR in the 3D spheroid forms.

scholarly journals Human endogenous retrovirus transcription profiles of the kidney and kidney-derived cell lines

2011 ◽  
Vol 92 (10) ◽  
pp. 2356-2366 ◽  
Author(s):  
Sonja Haupt ◽  
Michele Tisdale ◽  
Michelle Vincendeau ◽  
Mary Anne Clements ◽  
David T. Gauthier ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Cell Lines ◽  
Kidney Tissue ◽  
Human Kidney ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Human Endogenous Retrovirus ◽  
Tissue Samples ◽  
Transcription Pattern

The human genome comprises approximately 8–9 % of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to 22 RCC cell lines. No RCC cell line or tumour-specific differences were observed, suggesting that HERV transcription is not altered in RCC. However, when comparing tissue transcription to cell line transcription, there were consistent differences. The differences were irrespective of cancer state and included cell lines derived from non-tumour kidney tissue, suggesting that a specific alteration of HERV transcription occurs when establishing cell lines. In contrast to previous publications, all known HERV-derived tumour antigens, including those identified in RCC, were expressed both in multiple RCC cell lines and several non-tumour tissue-derived cell lines, a result that contrasts with findings from patient samples. The results establish the core kidney transcription pattern of HERVs and reveal differences between cell culture lines and tissue samples.

scholarly journals Isolation and identification of enteroviruses from sewage and sewage-contaminated water samples from Ibadan, Nigeria, 2012-2013

2017 ◽  
Author(s):  
Johnson Adekunle Adeniji ◽  
Adetunji Oladapo Adewale ◽  
Temitope Oluwasegun Cephas Faleye ◽  
Moses Olubusuyi Adewumi
Keyword(s):  
Cell Lines ◽  
Rna Extraction ◽  
Contaminated Water ◽  
Rt Pcr ◽  
Isolation And Identification ◽  
Wild Poliovirus ◽  
Southwest Nigeria ◽  
Species Specific ◽  
Mcf 7 ◽  
Ibadan Nigeria

AbstractIn 2010, we described sewage contaminated water (SCW) bodies that consistently yielded enteroviruses (EVs) in enterovirus surveillance (ES) sites in Lagos, Nigeria. By 2012, we demonstrated the presence and circulation of Wild Poliovirus 3 (WPV3) in these ES sites. Here we describe ES sites that consistently yield EVs in Ibadan metropolis southwest Nigeria.Twenty-five ES samples were collected by grab method from nine sites between October, 2012 and March, 2013. Samples were concentrated and four (RD, HEp2C, MCF-7 and L20B) different cell lines used for virus isolation from the concentrates. Isolates were subjected to RNA extraction, cDNA synthesis, PanEnterovirus 5l-UTR and VP1 assays. Unidentifiable isolates were further subjected to species-specific RT-PCR assays. Amplicons were sequenced, isolates identified and subjected to phylogenetic analysis.Twenty-five isolates were recovered from 8 (32%) of the 25 ES samples collected. Twenty-three of the isolates were identified as EVs by the PanEntero5l-UTR assay. Thirteen (57%) of the 23 EVs were positive for the VP1 assay, and identified as Coxsackievirus B3 (CVB3) (1 isolate), CVB6 (1 isolate), E6 (2 isolates), E7 (5 isolates), E11 (1 isolate), E12 (1 isolate) and E13 (2 isolates). None and 2 (25%) of the remaining isolates were positive for the EV-B and EV-C assays, respectively. The 2 EV-C positive enteroviruses were isolated on MCF-7.This study describes three very productive ES sites, and documents the presence of CVB3, CVB6, E6, E7, E11, E12 and, E13 in Ibadan, Nigeria. It shows that including other cell lines in EV isolation protocols can broaden the diversity of EV types recoverable.

scholarly journals Cell-Line Annotation on Europe PubMed Central

2014 ◽  
Author(s):  
Jee-Hyub Kim
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Biomedical Literature ◽  
Pubmed Central ◽  
Preliminary Results ◽  
Data Citation ◽  
A Cell

A cell line is a cell culture developed from a single cell and therefore consisting of cells with a uniform genetic make-up. A cell line has an important role as a research resource such as organisms, antibodies, constructs, knockdown reagents, etc. Unique identification of cell lines in the biomedical literature is important for the reproducibility of science. As data citation, resource citation is also important for resource re-use. In this paper, we mention the challenges of identifying cell lines and describe a system for cell line annotation with preliminary results.

scholarly journals Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture

Genetics ◽  
2021 ◽  
Author(s):  
Shunhua Han ◽  
Preston J Basting ◽  
Guilherme B Dias ◽  
Arthur Luhur ◽  
Andrew C Zelhof ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Loss Of Heterozygosity ◽  
Cell Lines ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
Drosophila Cell ◽  
Genome Wide ◽  
Cell Culture Systems ◽  
Cell Line Identity

Abstract Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabelling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of LTR retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.

scholarly journals Vaccineassociated paralytic poliomyelitis and acute flaccid paralysis on some territories of Russia during 20 years

2019 ◽  
Vol 11 (3) ◽  
pp. 102-109
Author(s):  
N. I. Romanenkova ◽  
N. R. Rozaeva ◽  
M. A. Bichurina ◽  
O. I. Kanaeva ◽  
I. G. Chkhyndzheriya
Keyword(s):  
Acute Flaccid Paralysis ◽  
Flaccid Paralysis ◽  
Paralytic Poliomyelitis ◽  
Nucleotide Substitutions ◽  
Isolation And Identification ◽  
Aim Analysis ◽  
Genome Region ◽  
Poliomyelitis Vaccine ◽  
Medical Recommendations ◽  
Type 3

Aim: Analysis of the morbidity of vaccine-associated paralytic poliomyelitis and acute flaccid paralysis and the results of virological investigation of the patients on 14 territories of the Russian Federation in 1998-2017. Materials and methods: We investigated nearly 3000 stool samples from paralytic patients and contact persons. Isolation and identification of polioviruses were performed according to WHO recommendations with the help of cell lines RD and L20B. We conducted the sequencing of the genome fragments VP3-VP1, VP1-2A and full sequencing of genome region VP1 of 45 poliovirus strains. Results: From 1998 till 2017 1257 cases of acute flaccid paralysis were registered on 14 territories of Russia, 15 cases of which (1,2%) were classified as vaccine-associated paralytic poliomyelitis. From these patients 9 children were non vaccinated and 6 children received from one to four doses of oral poliomyelitis vaccine. The percentage of the detection of polioviruses from the patients and contact persons in different years was not equal and constituted from 3, 4±0,89% to 9, 5±0,79%. All in all from the patients with acute flaccid paralysis and contact persons we isolated 191 polioviruses, 60 of them belonged to type 1, 55 polioviruses were identified as types 2 and 76 as type 3. Some cases of vaccine-associated paralytic poliomyelitis are described in the article; polioviruses were isolated from all these patients. The sequencing of the genome fragments of 45 poliovirus strains showed that the majority of them had the nucleotide substitutions including neurovirulent substitutions. Conclusion: In order to prevent the risk of the appearance of vaccine-associated paralytic poliomyelitis it is necessary to maintain the high quality of surveillance of poliomyelitis and acute flaccid paralysis, to ensure the 95% coverage of children with poliomyelitis vaccine, to minimize the cases of groundless delays of vaccination according to medical recommendations and parents’ refusals to vaccinate children against poliomyelitis and to respect strictly the National calendar of vaccination.

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