Scholarly works in 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 Towards an effective poliovirus laboratory containment strategy in Nigeria(BMC (BioMed Central),, 2018) Ticha, J. M.; Matthew, K. O.; Hamisu, A. W.; Braka, F.; Mkanda, P.; Nsubuga, P.; Tesfaye, E.; Craig, K,; Etsano, A.; Emelife, O.; Shuaib, F.; Akinkugbe, F.; Adeniji, J. A.; Adamu, U.; Dallatu, M.; Oyeyinka, G.; Brown, H.; Nnamah, N.; Okwori, J.; Chinedu, C.; Anibijuwon, I.; Adewumi, O. M.; Donbraye, E.; Bagana, M.; Baba, M.; Gumede, N.; Banda, R.; Tegegne, S. G.; Oyetunji, A.; Diop, O.; Tomori, O.; Vaz, R.G.Background: The Global Commission for the Certification of the Eradication of Poliomyelitis will declare the world free of wild poliovirus transmission when no wild virus has been found in at least 3 consecutive years, and all laboratories possessing wild poliovirus materials have adopted appropriate measures of containment. Nigeria has made progress towards poliomyelitis eradication with the latest reported WPV type 1 on 21 Aug 2016 after 2 years without any case. This milestone achievement was followed by an inventory of biomedical laboratories completed in November 2015 with the destruction of all identified infectious materials. This paper seeks to describe the poliovirus laboratory containment process in Nigeria on which an effective containment system has been built to minimize the risk of virus re-introduction into the population from the laboratories. Methods: A national survey of all biomedical facilities, as well as an inventory of laboratories from various sectors, was conducted from June November 2015. National Task Force (NTF) members and staff working on polio administered an on-site questionnaire in each facility. Laboratory personnel were sensitized with all un-needed materials destroyed by autoclaving and incineration. All stakeholders were also sensitized to continue the destruction of such materials as a requirement for phase one activities. Results: A total of 20,638 biomedical facilities were surveyed with 9575 having laboratories. Thirty laboratories were found to contain poliovirus or potentially infectious materials. The 30 laboratories belonged to the ministries of health, education, defence and private organizations. Conclusions: This article is amongst the first in Africa that relates poliovirus laboratory containment in the context of the tOPV-bOPV switch in alignment with the Global Action Plan III. All identified infectious materials were destroyed and personnel trained to continue to destroy subsequent materials, a process that needs meticulous monitoring to mitigate the risk of poliovirus re-introduction to the population.Item Draft genome sequence of a bovine enterovirus isolate recovered from sewage in Nigeria(American Society for Microbiology (ASM), 2018) Faleye, T. O. C; Adewumi, O. M.; Olayinka, O. A.; Donbraye, E; Oluremi, B.; George, U. E.; Arowolo, O. A.; Omoruyi, E. C.; Ifeorah, M. I.; Oyero, A . O.; Adeniji, J. A.We describe the draft genome of a bovine enterovirus (EV) isolate recovered from sewage in Nigeria. This isolate replicates on both RD and L20B cell lines but is negative for all EV screens in use by the Global Poliovirus Eradication Initiative (GPEI). It contains 7,368 nucleotides (nt) with 50.2% G C content and an open reading frame (ORF) with 6,525 nt (2,174 amino acids).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 Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria is geographically defined(Microbiology Society, 2018) Donbraye, E.; Olasunkanmi, O. I.; Opabode, B. A.; Ishola, T. R.; Faleye, T. O. C; Adewumi, O. M.; Adeniji, J. A.Purpose. We recently showed that enteroviruses (EVs) andenterovirus species C (EV-C) in particular were abundant in faecal samples from children who had been 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 varies by geographical region. Methodology. One hundred and eight samples (i.e. 54 paired stool suspensions from 54 AFP cases) that had previously been confirmed to be negative for EVs by the WHO recommended RD-L20B cell culture-based algorithm were analysed. The 108 samples were made into 54 pools (27 each from North-West and South-South Nigeria). All were subjected to RNA extraction, cDNA synthesis and the WHO-recommended semi-nested PCR assay and its modifications. All of the amplicons were sequenced, and the enteroviruses identified, using the enterovirus genotyping tool and phylogenetic analysis. Results. EVs were detected in 16 (29.63 %) of the 54 samples that were screened and successfully identified in 14 (25.93 %). Of these, 10 were from North-West and 4 were from South-South Nigeria. One (7.14 %), 2 (14.29 %) and 11 (78.57 %) of thestrains detected were EV-A, EV-B and EV-C, respectively. The 10 strains from North-West Nigeria included 7 EV types, namely CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from South-South Nigeria were E31, CV-A1, EV-C99 and EV-C116. Conclusion. The results of this study showed that the presence of EVs and consequently EV-Cs in AFP samples declared to be EV-free by the RD-L20B cell culture-based algorithm varies by geographical region in Nigeria.Item Recovery of nonpolio enteroviruses from l20b cell line with non-reproducible cytopathic effect(BioMed Central, 2018) Adeniji, J. A.; Ibok, U. I.; Ayinde,O. T.; Oragwa, A. O.; George, U. E.; Faleye. T. O. C.; Adewumi, M. O.Background and Aim of the Study: Samples showing cytopathic effect (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. Place and Duration of Study: This study was carried out in the Department of Virology, College of Medicine, University of Ibadan using twenty-six (26) cell culture suspensions 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 faecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. The study lasted for three (3) months from samples collection to report writing. Methodology: All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminested PCR assay, species resolution PCR assay, sequencing andphylogenetic analysis. Results: 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 (1isolate). Conclusion: 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.Item Extending the utility of the WHO recommended assay for direct detection of enteroviruses from clinical specimen for resolving poliovirus co‑infection(BMC Research Notes, 2018) Faleye, T. O. C; Adewumi, M. O.; Ozegbe, N. P.; Ogunsakin, O. E.; Ariyo, G.; Adeshina, F. W.; Ogunga, O. S.; Oluwadare, S. D.; Adeniji, J. A.Objectives: In a polio-free world there might be reduced funding for poliovirus surveillance. There is therefore the need to ensure that enterovirologist globally, especially those outside the global polio laboratory network, can participate in poliovirus surveillance without neglecting their enterovirus type of interest. To accomplish this, assays are needed that allow such active participation. Results: In this study we describes a sensitive and specific utility extension of the recently recommended WHO RT-snPCR assay that enables independent detection of the three poliovirus types especially in cases of co-infection. More importantly, it piggy-backs on the first round PCR product of the WHO recommended assay and consequently ensures that enterovirologists interested in nonpolio enteroviruses can continue their investigations, and contribute significantly and specifically to poliovirus surveillance, by using the excess of their first round PCR product.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.
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