Virology
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Item Isolation and Complete Capsid Sequence of Enterovirus D111 from Faeces of a Child with Acute Flaccid Paralysis in Nigeria(ResearchersLinks Ltd, 2019) Faleye, T. O. C.; Adewumi, M. O.; Olayinka, O. T.; Adeniji, J. A.The 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 cell culture supernatants (CCS) (collected between 2016 and 2017) from RD and L20b cell-culture-tubes with NR-CPE, were analyzed. 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 (EAVs) and group A Rotavirus (GARV) 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 EAVs and GARVs. Four CCS produced R-CPE in HEp-2 cell line, three of which were positive for the 5l-UTR assay. Of the three 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. 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-D isolates of Nigerian origin and the first complete capsid sequence of the virus globally. We also show that NR-CPE could sometimes be caused by EVs that do not produce R-CPE in specific cell lines.Item Characterization of hepatitis delta virus strains spreading in Abuja, Nigeria(Wiley Periodicals, Inc.,, 2019) Ifeorah, I. M.; Faleye, T. O. C.; Bakarey, A. S.; Adewumi, O. M.; Gerber, A.; Le Gal, F.; Adeniji, J. A; Gordien, E.; Onyemelukwe, N. F.Hepatitis delta virus (HDV) is responsible for the most severe form of liver disease in humans. So far, eight genotypes (HDV‐1 to ‐8) have been individualized worldwide. Little is known about HDV strains that spread in Nigeria. HDV genotyping was performed in 15 anti–HDV positive samples from a cohort of 306 hepatitis B virus (HBV)‐infected patients in Abuja (Nigeria). Phylogenetic analyses revealed 90% were HDV‐1, two among them clustering with European/Asian HDV‐1, the remaining one being HDV‐6. It was also found that two members of a couple superinfected with the same HDV strain, were enveloped by two different HBV strains of genotype E.Item Reference Echovirus 7 and 19 Genomes from Nigeria(American Society for Microbiology (ASM), 2018) Adeniji, J. A.; Faleye, T. O. C.; Adewumi, O. M.We describe the genomes of two echovirus isolates from Nigeria as reference enterovirus species B genomes for the region. These echovirus 7 and 19 genomes have 7,411 nucleotides (nt) and 7,426 nt and were recovered from sewagecontaminated water (in 2010) and an acute flaccid paralysis case (in 2014), respectively.Item Identification of previously untypable RD cell line isolates and detection of EV-A71 genotype C1 in a child with AFP in Nigeria(Taylor & Francis, 2018-11-26) Adewumi, O. M.; Faleye, T. O. C.; Okeowo, C. O.; Oladapo, A. M.; Oyathelemhi, J.; Olaniyi, O. A.; Isola, O. C.; Adeniji, J. A.We previously attempted to identify 96 nonpolio enteroviruses (EVs) recovered in RD cell culture from children <15years with acute flaccid paralysis in Nigeria. We succeeded in identifying 69 of the isolates. Here, we describe an attempt to identify the remaining 27 isolates. Twenty-six (the 27th isolate was exhausted) isolates/samples that could not be typed previously were further analyzed. All were subjected to RNA extraction, cDNA synthesis, enterovirus 5ʹ-UTR–VP2 PCR assay and a modified VP1 snPCR assay. Both the 5ʹ-UTR–VP2 and VP1 amplicons were sequenced, isolates identified and subjected to phylogenetic analysis. Twenty of the 26 samples analyzed were identified. Altogether, 23 (three samples had co-infection) EV strains were recovered. These belong 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 (1strain) and EVC99 (2 strains). Of the 11 EV types, the 5ʹ-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. We successfully identified 20 of 26 samples that were previously untypable. We also provided evidence that suggests a clade of EVA71 genotype C1 might have been circulating in sub-Saharan Africa since 2008. Finally, we showed that the 5ʹ-UTR -VP2 assay might be as valuable as the VP1 assay in EV identification.Item Draft Genome Sequence of Mycoplasma arginini Strain NGR_2017(Merican Society for Microbiology (ASM), 2018-06-28) Adeniji, J.A.; Faleye, T. O. C.; Adewu , M. O.; Olayinka O. A.; Donbraye, E.; Oluremi, B.; George,a U. E.; Arowolo, O. A.; Omoruy, E. C.; Ifeorah, M. I.; Akande, A.We present the draft genome of Mycoplasma arginini strain NGR_2017. This strain was recovered in Nigeria from cell culture in 2017. The assembly contains 620,555 bp in 12 contigs. It contains 561 coding sequences, 34 RNAs (29 tRNAs, 4rRNAs, and 1 transfer-messenger RNA [tmRNA]), and a_26-kb integrative and conjugative element.Item Evaluation of CD4 T lymphocyte cell Levels among Hepatitis B, C and E Viruses negative individuals in Ibadan, Southwestern Nigeria(SCIENCEDOMAIN International, 2017) Adewumi, M. O .; Omoruyi, E. C.; Ifeorah, I. M.; Bakarey, A. S.; Ogunwale, A. O.; Akere, A.; Faleye, T. O. C.; Adeniji, J.A.Aim: The CD4 T lymphocytes play a key role in achieving a regulated effective immune response to foreign antigens. It is also a valuable parameter for assessing HIV disease progression. However, variations in CD4 T lymphocyte values due to diverse factors have been reported. Here we evaluated CD4 T lymphocytes among community dwellers who tested negative for hepatitis B, hepatitis C and hepatitis E viruses and compared the results with the National Reference Values (NRVs).Study Design: A cross-sectional study was conducted. Participants were enrolled using a convenient sampling technique and their socio-demographic characteristics were captured by administration of semi-structured questionnaires. Place and Duration of Study: This study was conducted among residents of Ibadan metropolis, Southwestern Nigeria. Participants were enrolled between July and September, 2013 at the University College Hospital, Ibadan. Methodology: Four hundred consenting participants who fulfilled the criteria for enrolment were evaluated for CD4 T lymphocyte counts. Results: Estimated mean CD4 T lymphocyte count of 1,183 (CD4 Range: 328-2680) cells/μl of blood was recorded for the participants. Four (1.0%), 151 (37.8%), 157 (39.2%), 74 (18.5), and 14(3.5) of the participants had CD4 T lymphocyte count ranged 352-500, 501-1,000, 1,001-1500, 1501-2,000, and >2,000 cells/μl of blood, respectively. Differences in the estimated mean CD4 count between different age groups varied significantly (P=0.010).Conclusion: In this study, significantly higher CD4 T lymphocyte values were observed among the study population in comparison to the NRVs, and consequently we advise careful interpretation and use of extrapolated CD4 T lymphocyte values in the management of persons with diverse geographical background or health conditions.Item The fallacy of identification by neutralization in the light of non-cytopathic effect producing enterovirus strains(SCIENCEDOMAIN International, 2017) Adewumi, M. O.; Faleye, T. O. C.; Adeniji, J. A.We describe the characterization of an enterovirus isolate recovered from untreated raw sewage in Ibadan, southwest Nigeria in 2010. The isolate was neutralized by specific antisera and consequently identified as Echovirus 7 (E7). Subsequent molecular characterization showed the isolate to be a mixture of E7 and Coxsackievirus A24 (CV-A24) thereby suggesting the CV-A24 strain to be non-cytopathic effect producing. It is therefore crucial that identities of enterovirus strains determined by neutralization assay be verified by molecular characterization to ensure they do not have any non-cytopathic effect producing strain(s) lurking unnoticed.Item Comparison of Algorithms for the Detection of Enteroviruses in Stool Specimens from Children Diagnosed with Acute Flaccid Paralysis(Hindawi Publishing Corporation, 2017) Adeniji, J. A.; Ayeni, F. A.; Ibrahim, A.; Tijani, K. A.; Faleye, T. O. C.; Adewumi, M. O.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.Item Isolation and Identification of Enteroviruses from Sewage and Sewage-Contaminated Water Samples from Ibadan, Nigeria, 2012-2013(SciTechnol, 2017) Adeniji, J. A.; Adewale, A. O.; Faleye, T. O. C.; Adewumi, M. O.In 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 RTPCR assays. Amplicons were sequenced, isolates identified and subjected to phylogenetic analysis. Twenty-five isolates were recovered from 8 (32%) of the 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. Including other cell lines in EV isolation protocols can broaden the diversity of EV types recoverable.Item Development of Echovirus 29 Cytopathology in RD Cell Line Might not Happen within 10 Days Post Inoculation(SciTechnol, 2017) Adewumi, M. O.; Faleye, T. O. C.; Ayinde, O. T.; Ibok, U. I.; Adeniji, J. A.Classically in enterovirology, isolation in RD cell culture required inoculation and incubation for 14 days before any sample is declared negative for enteroviruses. To reduce the turn-around time for confirming suspected cases of poliomyelitis, in 2010, the required incubation period, post inoculation was reduced to 10days. In this study we set out to determine why an Echovirus 29 (E29) strain we isolated in 2016 showed no cytopathology on RDcell culture despite the fact that other members of the clade to which it belongs do so. We found that the problem was not the 2016 E29 strain but the new 10 days incubation algorithm. To be precise, the 2016 E29 strain did not show cytopathology in RD cell line within the recommended 10 days but replicated with evident cytopathology when allowed to stay in culture for 13 to 14 days. This shows that some samples declared negative for enteroviruses by the 10 days incubation algorithm might be false negatives that will eventually develop cytopathology if allowed to remain in incubation for 13 to 14 days. It is therefore encouraged that those particularly interested in Non-Polio Enteroviruses endeavour to maintain at least 14 days incubation in cell culture in a bid to accommodate the likes of E29 that might need 13-14 days to develop CPE especially when present at low titre.