High Diversity of Human Non-Polio Enterovirus Serotypes Identified in Contaminated Water in Nigeria.

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017-2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.

Identification of previously untypable RD cell line isolates and detection of EV-A71 genotype C1 in a child with AFP in Nigeria.

We previously attempted to identify 96 nonpolio enteroviruses (EVs) recovered in RD cell culture from children <15 years 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 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 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 (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. 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 5'-UTR -VP2 assay might be as valuable as the VP1 assay in EV identification.

Abundance of enterovirus C in RD-L20B cell culture-negative stool samples from acute flaccid paralysis cases in Nigeria is geographically defined.

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 the strains 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.

Extending the utility of the WHO recommended assay for direct detection of enteroviruses from clinical specimen for resolving poliovirus co-infection.

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.

Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay.

Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

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.

Enterovirus Species B Bias of RD Cell Line and Its Influence on Enterovirus Diversity Landscape.

Despite its widespread use in poliovirus isolation, studies show that most RD cell line isolates are species B enteroviruses (EB), it was therefore employed to further catalogue the EB diversity in two different regions of Nigeria. Concentrates of 18 environmental samples were inoculated into RD cell line. Isolates were subjected to PCR assays to detect enteroviruses, species C and B members and partial VP1 gene which was subsequently sequenced and used for identification and phylogenetic analysis. Isolates were further passaged in L20B cell line to detect polioviruses. Sixty-eight isolates were recovered from the 18 concentrates, all of which were positive for the enterovirus 5'-UTR screen. Thirteen of the 68 isolates were positive for the species C screen and replicated in L20B cell line, eleven of which also contained species B enteroviruses. Some of the mixed isolates were successfully typed, but as species B members. In all, isolates recovered in this study were identified as CVB5, E6, E7, E11, E13, E19, E20, E33, EVB75 and WPV3, while some could not be typed. Alongside the ten different enterovirus serotypes confirmed, results of this study document for the first time in Nigeria, EVB75. It showed the EB bias of RD cell line might indicate something much more fundamental in its biology. Finally, the finding of WPV3 in a region considered low risk for poliovirus emphasizes the need to expand poliovirus environmental surveillance to enable early detection of poliovirus silent circulation before occurrence of clinical manifestations.

Detection and circulation of hepatitis B virus immune escape mutants among asymptomatic community dwellers in Ibadan, southwestern Nigeria.

BACKGROUND: In 2012, the first Nigerian Hepatitis B Virus (HBV) immune escape mutant (IEM) case was detected in a pregnant woman in southwestern Nigeria. Consequently, this study was designed to investigate the presence and possible circulation of IEMs amongst asymptomatic community dwellers in southwestern Nigeria. METHODS: Blood specimens collected from 438 asymptomatic community dwellers were screened for HBsAg using ELISA technique. Subsequently, the S-gene was amplified in HBsAg positive samples by a nested PCR protocol, and amplicons sequenced. Isolates were then subtyped by amino acid residues at positions 122, 127, 134 and 160, and genotyped by phylogenetic analysis. RESULTS: Of the 31 (7.08%) samples positive for HBsAg, the ∼ 408 bp Sgene fragment was successfully amplified and sequenced in 27. Samples obtained from 4 patients could not be amplified due to low titres. Sequence data from only 15 of the isolates could be analysed further as eight of the remaining 12 had multiple peaks while the rest three showed no similarity to any HBV gene when subjected to BLAST analysis. Thirteen of the 15 isolates were identified as genotype E. Eleven of which were subtyped as ayw4 while the remaining two could not be subtyped due to sR122Q/P substitutions. The last two isolates that could not be genotyped and subtyped had other mutations in the "a" determinant associated with IEMs. CONCLUSIONS: This study confirmed presence and circulation of HBV IEM in Nigeria, the country's inclusion in the genotype E crescent, and the value of phylogenetic analysis in HBV identification.

Detection of hepatitis B virus isolates with mutations associated with immune escape mutants among pregnant women in Ibadan, southwestern Nigeria.

Perinatal transmission of hepatitis B virus (HBV) and its associated immune escape mutants (IEMs), is the major vehicle through which a population of chronically infected people who serve as infectious HBV reservoirs is maintained in communities. Therefore, to assess the risk of perinatal transmission, 272 pregnant women attending ante-natal clinics in Ibadan metropolis, southwestern, Nigeria, were screened for HBsAg using ELISA technique. Samples positive for HBsAg were subjected to HBV DNA detection by PCR amplification of the S-gene and amplicon sequencing. Isolates were genotyped and subtyped using a combination of molecular techniques. Fifteen (5.5%) of the pregnant women were positive for HBsAg of which HBV DNA was detected in seven. Five of the isolates were typed as genotype E subtype ayw4 using amino acid residues at positions 122, 127, 134 and 160. Another could only be typed as genotype E subtype ayw4 by further phylogenetic analysis. The remaining one isolate did not belong to any of genotypes A - H. Three of the HBV isolates including the untypable, had mutations in the 'a' determinant associated with IEMs. This study confirms the endemicity of HBV, the risk of perinatal transmission and the circulation of genotype E subtype ayw4 in Nigeria. It further demonstrates the presence of IEMs in Nigeria.

Enterovirus C strains circulating in Nigeria and their contribution to the emergence of recombinant circulating vaccine-derived polioviruses.

Between 2005 and 2011, 23 lineages of circulating vaccine-derived polioviruses (cVDPVs) were detected in Nigeria with nonstructural region (NSR) of non-polio enterovirus C (NPEV-C) origin. However, no information exists on NPEV-C strains recombining with oral poliovirus type 2 vaccine strains (OPV2) to make type 2 cVDPVs (cVDPV2s) in Nigeria. This study was therefore designed to investigate the probable contribution of NPEV-Cs recently isolated in the region to the emergence of cVDPV2s. Eleven enterovirus C (EV-C) strains (8 NPEV-Cs and 3 PV2s) previously isolated by the authors were analysed in this study. All 11 isolates were assayed for cell-line-dependent growth restriction in four cell lines (LLC-MK2, MCF-7, RD and L20B). Subsequently, the isolates were subjected to RT-PCR specific for VP1 and 3Dpol/3'-UTR of EV-C. All PCR products were sequenced, and phylogenetic analysis was performed. All eight NPEV-Cs replicated exclusively in the MCF-7 cell line, while the three PV2s replicated in all four cell lines. The eight NPEV-Cs were identified as CVA13 (7 isolates) and CVA20 (1 isolate) by VP1 analysis, while all 11 isolates were confirmed to be EV-Cs by 3Dpol/3'-UTR analysis. In addition, phylogeny violations suggested that some cVDPVs might have recombined with common ancestors of the NPEV-Cs described in this study. This was confirmed by the scatter plot of divergence in VP1 against that of 3Dpol/3'-UTR sequences for pairs of isolates. The results of this study showed that the NSR of unknown origin found in cVDPVs from the region might have come from NPEV-Cs (e.g., CVA13 and CVA20) circulating in Nigeria.

Impact of cell lines included in enterovirus isolation protocol on perception of nonpolio enterovirus species C diversity.

There has been under-reporting of nonpolio enterovirus species Cs (NPESCs) in Nigeria despite the fact that most isolates recovered from the Nigerian vaccine derived poliovirus serotype 2 (VDPV2) outbreak were recombinants with nonstructural region of NPESC origin. It has been suggested that cell lines included in enterovirus isolation protocols might account for this phenomenon and this study examined this suggestion. Fifteen environmental samples concentrated previously and analysed using L20B and RD cell lines as part of the poliovirus environmental surveillance (ES) program in Nigeria were randomly selected and inoculated into two cell lines (MCF-7 and LLC-MK2). Isolates were identified as enteroviruses and species C members using different RT-PCR assays, culture in L20B cell line and sequencing of partial VP1. Forty-eight (48) isolates were recovered from the 15 samples, 47 (97.9%) of which were enteroviruses. Of the enteroviruses, 32 (68.1%) belonged to enterovirus species C (EC) of which 19 (40.4%) were polioviruses and 13 (27.7%) were NPESC members. All 13 NPESC isolates were recovered on MCF-7. Results of the study show that NPESCs are circulating in Nigeria and their under-reporting was due to the combination of cell lines used for enterovirus isolation in previous reports.

Isolation and identification of enteroviruses from sewage and sewage-contaminated water in Lagos, Nigeria.

Studies have confirmed silent circulation of enteroviruses in the environment even in the absence of associated clinical conditions in the community. In this light, 26 samples of sewage and sewage-contaminated water serving selected high-risk communities in Lagos Nigeria were examined between June and September 2010. To concentrate virus particles in the sample, 480 µL of each sample was centrifuged at 3,000 rpm for 1 h at 4 °C. Subsequently, pellets were pooled, chloroform treated and further centrifuged at 1,500 rpm for 20 min at 4 °C. The water phase (concentrate) was then collected and stored at -20 °C. The concentrates were subsequently inoculated into RD and L20B cell lines. Recovered isolates were identified by real-time RT-PCR (rRT-PCR), serotyping, VP1 amplification, sequencing and phylogenetic analysis. Overall, 9 (34.6%) of the samples showed characteristic enterovirus cytopathic effect in RD cell line and were subsequently confirmed by pan-enterovirus rRT-PCR. The isolates were further identified by serotyping to include three E7, one E11 and one E13 isolates whilst four isolates were untypable. Further characterisation by VP1 sequencing confirmed the results of serotyping and rRT-PCR for all but isolate E13. Also, the four previously untypable isolates were identified to include two E19, one E20 and one E7 by VP1 sequencing. Results of the study confirmed circulation of Sub-Saharan Africa-specific enterovirus clades in the region, provide information on their molecular epidemiology and emphasise the need to combine methods of identification to enhance enterovirus surveillance.