Virology

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    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.
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    "Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?"
    (MDPI, 2015) Faleye, T. O. C.; Adewumi, M. O.; Adeniji, J. A.
    Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay.More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape.