Johnson Adekunle Adeniji

High-Throughput Next-Generation Sequencing of Polioviruses

ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance.

Sabin Vaccine Reversion in the Field: a Comprehensive Analysis of Sabin-Like Poliovirus Isolates in Nigeria.

ABSTRACT To assess the dynamics of genetic reversion of live poliovirus vaccine in humans, we studied molecular evolution in Sabin-like poliovirus isolates from Nigerian acute flaccid paralysis cases obtained from routine surveillance. We employed a novel modeling approach to infer substitution and recombination rates from whole-genome sequences and information about poliovirus infection dynamics and the individual vaccination history. We confirmed observations from a recent vaccine trial that VP1 substitution rates are increased for Sabin-like isolates relative to the rate for the wild type due to increased nonsynonymous substitution rates. We also inferred substitution rates for attenuating nucleotides and confirmed that reversion can occur in days to weeks after vaccination. We combine our observations for Sabin-like virus evolution with the molecular clock for VP1 of circulating wild-type strains to infer that the mean time from the initiating vaccine dose to the earliest detection of circulating vaccine-derived poliovirus (cVDPV) is 300 days for Sabin-like virus type 1, 210 days for Sabin-like virus type 2, and 390 days for Sabin-like virus type 3. Phylogenetic relationships indicated transient local transmission of Sabin-like virus type 3 and, possibly, Sabin-like virus type 1 during periods of low wild polio incidence. Comparison of Sabin-like virus recombinants with known Nigerian vaccine-derived poliovirus recombinants shows that while recombination with non-Sabin enteroviruses is associated with cVDPV, the recombination rates are similar for Sabin isolate-Sabin isolate and Sabin isolate–non-Sabin enterovirus recombination after accounting for the time from dosing to the time of detection. Our study provides a comprehensive picture of the evolutionary dynamics of the oral polio vaccine in the field. IMPORTANCE The global polio eradication effort has completed its 26th year. Despite success in eliminating wild poliovirus from most of the world, polio persists in populations where logistical, social, and political factors have not allowed vaccination programs of sustained high quality. One issue of critical importance is eliminating circulating vaccine-derived polioviruses (cVDPVs) that have properties indistinguishable from those of wild poliovirus and can cause paralytic disease. cVDPV emerges due to the genetic instability of the Sabin viruses used in the oral polio vaccine (OPV) in populations that have low levels of immunity to poliovirus. However, the dynamics responsible are incompletely understood because it has historically been difficult to gather and interpret data about evolution of the Sabin viruses used in OPV in regions where cVDPV has occurred. This study is the first to combine whole-genome sequencing of poliovirus isolates collected during routine surveillance with knowledge about the intrahost dynamics of poliovirus to provide quantitative insight into polio vaccine evolution in the field.

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.

Some genetic characteristics of sabin-like poliovirus isolated from acute flaccid paralysis cases in Nigeria

A total of 34 sabin strains of the poliovirus isolated from 22 children with 60-day follow-up residual acute flaccid paralysis (AFP) were genetically characterized and screened for any form of recombination. Sequence analysis of the 906-nucleotide capsid showed that all the isolates were similar to their original sabin serotypes, however two of the viruses had drifted in their 3D noncapsid regions toward a sabin-sabin and sabin-nonpolio entero combination. Routine immunization in Nigeria is low and in spite of the increase in the frequency of supplemental immunizations, a lot of children are still inadequately immunized, which may be the reason for our observation in this study. Although we are not dealing with a case of circulating vaccine derived poliovirus (cVDPV) yet, if the above condition persists, the advent of cVDVP may not be too far. There is therefore the need to maintain a high quality mass immunization and sustained routine immunization. Key words : Poliovirus, sequence, crossover, non polio enterovirus, recombination, genome, Sabin-like, vaccine, Nigeria. African Journal of Biotechnology Vol.2(11) 2003: 460-464

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.

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 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.

Contribution of Environmental Surveillance Toward Interruption of Poliovirus Transmission in Nigeria, 2012–2015

Background. Cases of paralysis caused by poliovirus have decreased by >99% since the 1988 World Health Assemblys resolution to eradicate polio. The World Health Organization identified environmental surveillance (ES) of poliovirus in the poliomyelitis eradication strategic plan as an activity that can complement acute flaccid paralysis (AFP) surveillance. This article summarizes key public health interventions that followed the isolation of polioviruses from ES between 2012 and 2015. Methods. The grap method was used to collect 1.75 L of raw flowing sewage every 2–4 weeks. Once collected, samples were shipped at 4°C to a polio laboratory for concentration. ES data were then used to guide program implementation. Results. From 2012 to 2015, ES reported 97 circulating vaccine-derived polioviruses (cVDPV2) and 14 wild polioviruses. In 2014 alone, 54 cVDPV type 2 cases and 1 WPV type 1 case were reported. In Sokoto State, 58 cases of AFP were found from a search of 9426 households. A total of 2 252 059 inactivated polio vaccine and 2 460 124 oral polio vaccine doses were administered to children aged <5 year in Borno and Yobe states. Conclusions. This article is among the first from Africa that relates ES findings to key public health interventions (mass immunization campaigns, inactivated polio vaccine introduction, and strengthening of AFP surveillance) that have contributed to the interruption of poliovirus transmission in Nigeria.

Preponderance of enterovirus C in RD-L20B-cell-culture-negative stool samples from children diagnosed with acute flaccid paralysis in Nigeria

Recently, a reverse transcriptase semi-nested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses in clinical specimens. In this study, we use this assay and a modification thereof to screen acute flaccid paralysis (AFP) samples that had previously tested negative for enteroviruses by the RD-L20B algorithm. Thirty paired stool suspensions collected in 2015 as part of the national AFP surveillance program in different states of Nigeria were analyzed in this study. The samples had previously tested negative for enteroviruses in the polio laboratory in accordance with the WHO-recommended RD-L20B-cell-culture-based algorithm. Two samples that had previously been found to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, the RT-snPCR assay and a modified version of the RT-snPCR. All amplicons were sequenced, and enteroviruses were identified using the enterovirus genotyping tool and phylogenetic analysis. Amplicons were recovered from the two controls and 50% (15/30) of the samples screened. Fourteen were successfully typed, of which, 7.1% (1/14), 21.4% (3/14), 64.3% (9/14) and 7.1% (1/14) were enterovirus (EV) -A, EV-B, EV-C and a mixture of EV-B and C (EV-C99 and E25), respectively. The two controls were identified as EV-C99 and coxsackievirus (CV) -A1, both of which belong to the species Enterovirus C. In one sample, poliovirus serotype 2 was detected and found to have the VP1 ILE143 variation and was therefore identified as a vaccine strain. The results of this study showed that significant proportion of enterovirus infections (including some with Sabin PV2) are being missed by the RD-L20B-cell-culture-based algorithm, thus highlighting the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-C in Nigeria was also confirmed.