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

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.

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 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.

A REVISED GENERIC SYNOPSIS OF HYACINTHACEAE IN SUB-SAHARAN AFRICA, BASED ON MOLECULAR EVIDENCE, INCLUDING NEW COMBINATIONS AND THE NEW TRIBE PSEUDOPROSPEREAE

2003 ◽  
Vol 60 (3) ◽  
pp. 533-568 ◽  
Author(s):  
J. C. MANNING ◽  
P. GOLDBLATT ◽  
M. F. FAY
Keyword(s):  
Phylogenetic Analysis ◽  
Sub Saharan Africa ◽  
Molecular Evidence ◽  
New Combinations ◽  
Generic Level ◽  
The Family ◽  
Molecular Phylogenetic ◽  
Single Genus ◽  
New Tribe ◽  
Sub Saharan

A revised generic synopsis of sub-Saharan Hyacinthaceae is presented, based on a molecular phylogenetic analysis of the family. Generic rank is accorded only to reciprocally monophyletic clades that can be distinguished by recognizable morphological discontinuities, thereby permitting an appropriate generic assignment of species not included in the analysis. Three subfamilies are recognized within the region. Subfamily Ornithogaloideae, characterized by flattened or angular seeds with tightly adhering testa, is considered to include the single genus Ornithogalum, which is expanded to include the genera Albuca, Dipcadi, Galtonia, Neopatersonia and Pseudogaltonia. Recognizing any of these segregates at generic level renders the genus Ornithogalum polyphyletic, while subdivision of Ornithogalum into smaller, morphologically distinguishable segregates in order to preserve the monophyly of each is not possible. Subfamily Urgineoideae, characterized by flattened or winged seeds with brittle, loosely adhering testa, comprises the two mainland African genera Bowiea and Drimia. The latter is well circumscribed by its deciduous, short-lived perianth and includes the previously recognized genera Litanthus, Rhadamanthus, Schizobasis and Tenicroa. The monotypic Madagascan Igidia is provisionally included in the subfamily as a third genus on the basis of its seeds, pending molecular confirmation of its relationships. Subfamily Hyacinthoideae resolves into three clades, distinguished as tribes Hyacintheae (strictly northern hemisphere and not treated further), Massonieae and Pseudoprospereae tribus nov. Full descriptions and a key to their identification are provided for all genera. New combinations reflecting the generic circumscriptions adopted here are made for most African and all Indian and Madagascan species.

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

scholarly journals Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 201 ◽  
Author(s):  
Alexander Falkenhagen ◽  
Corinna Patzina-Mehling ◽  
Ashish K. Gadicherla ◽  
Amy Strydom ◽  
Hester G. O’Neill ◽  
...  
Keyword(s):  
Cell Culture ◽  
Disease Burden ◽  
Reverse Genetics ◽  
Sub Saharan Africa ◽  
Rotavirus A ◽  
Virus Particles ◽  
Rotavirus Vaccines ◽  
Human Rotavirus ◽  
Sub Saharan ◽  
Infants And Young Children

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.

scholarly journals Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

2004 ◽  
Vol 70 (3) ◽  
pp. 1483-1486 ◽  
Author(s):  
Hui Wang ◽  
Fanrong Kong ◽  
Peter Jelfs ◽  
Gregory James ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Cell Culture ◽  
16S Rrna ◽  
Cell Line ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Pcr Assay ◽  
Intergenic Spacer Region ◽  
Specific Pcr ◽  
Specific Pcr Assay ◽  
Species Specific

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

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