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 Rapid HIV Antigen–Antibody Assays and detection of acute HIV infection in sub-saharan Africa(American Society of Tropical Medicine and Hygiene, 2019) Adetunji, A. A.; Adewumi, M. O.; Michael, O. S.; Fayemiwo, S. A.; Ogunniyi, A.; Taiwo, B. O.Detection of acute HIV infection is a unique problem that fourth-generation HIV assays were expected to alleviate. In this commentary, we draw attention to the limitations and challenges with use of currently available rapid antigen–antibody (Ag/Ab) combination tests for detection of acute HIV infection in sub-Saharan Africa. Laboratory-based HIV-1 Ag/Ab immunoassays are complex, requiring specialized equipment and handling that are currently not affordable in many settings in Africa. The point-of-care Ag/Ab platform on the other hand is easier to deploy and potentially more accessible in resource-limited settings. However, available fourth-generation HIV-1 rapid diagnostic tests have demonstrated poor performance characteristics in field studies where non-B subtypes of HIV-1 dominate. The potential for point-of-care HIV-1 Ag/Ab diagnostics to significantly improve detection of acute HIV infection remains yet to be realized in sub-Saharan Africa. Assay platforms need to be optimized to identify local circulating subtypes, and optimal algorithms need to be determinedItem 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.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.Item Profiles of Hepatitis B Virus Serological Markers among Asymptomatic Population in Anambra State, Southeastern Nigeria(SciTechnol, 2017) Bakarey, A. S.; Ifeorah, I. M.; Adewumi, M. O.; Faleye, T. O. C.; Akere, A.; Omoruyi, C. E.; Ogunwale, A. O.; Uttah, C. C.; Oketade, M. A.; Adeniji, J. A.Hepatitis B Virus (HBV) infection is apparent in endemic countries affecting millions of people. Further, the asymptomatic nature of the pathogen is a major public health concern. This study was designed to assess the burden of HBV by exploring the serologic markers of infection among consenting asymptomatic community dwellers in two cities in southeastern Nigeria. A total of 405 blood specimens were tested for HBsAg, anti-HBs, HBeAg, anti-HBe, total anti-HBc and anti-HBc-IgM using ELISA technique. Overall, 14(3.5%) of the participants had detectable HBsAg out of which 1 (7.1%) had HBeAg and 13, anti-HBe. Two of the HBsAg positives (14.3%) had detectable anti-HBc-IgM. A total of 144 (35.5%) had detectable anti-HBc, even as 65 (57.0%) of them had the marker as the only serologic evidence of HBV exposure. Thirty-seven (9.1%) participants had anti-HBs only although all of them were born before the start of the childhood HBV vaccination. Altogether, 224 (57.3%) had no detectable serological markers of HBV infection or immunity and were obviously at risk ofHBV infection. This study described various patterns of HBV serologic markers of infection in the study population and probable risk of viru spread. Our results support the need for urgent intervention and implementation of measures to control the spread of HBV infection in Nigeria.Item Acute Hepatitis E Virus Infection in Two Geographical Regions of Nigeria(Hindawi Publishing Corporation, 2017) Ifeorah, I. M.; Bakarey, A. S.; Faleye, T. O. C.; Adewumi, M. O.; Akere, A.; Omoruyi, C. E.; Ogunwale, A. O.; Awokunle, R. F.; Sekoni, D. E.; Adeniji, J. A.This study was therefore designed to describe acute HEV infection among antenatal clinic attendees and community dwellers from two geographical regions in Nigeria. Seven hundred and fifty plasma samples were tested for HEV IgM by Enzyme Linked Immunosorbent Assay (ELISA) technique. The tested samples were randomly selected from a pool of 1,115 blood specimens previously collected for viral hepatitis studies among selected populations (pregnant women, 272; Oyo community dwellers, 438; Anambra community dwellers, 405) between September 2012 and August 2013. One (0.4%) pregnant woman in her 3rd trimester had detectable HEV IgM, while community dwellers fromthe two study locations had zero prevalence rates of HEV IgM.Detection of HEV IgM in a pregnant woman, especially in her 3rd trimester, is of clinical and epidemiological significance.The need therefore exists for establishment of a robust HEV surveillance system in Nigeria and especially amidst the pregnant population in a bid to improve maternal and child health.