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 143 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.

AbstractsRecently, a reverse transcriptase seminested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses from clinical specimen. In this study, we use the assay and its modification to screen acute flaccid paralysis (AFP) samples previously confirmed 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 were previously confirmed negative for enteroviruses by the polio laboratory in accordance with the WHO recommended RD-L20B cell culture based algorithm. Two samples previously confirmed to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, and the RT-snPCR assay and its modifications. All amplicons were sequenced and enteroviruses identified using the enterovirus genotyping tool.Overall, amplicons were recovered from the two controls and 50% (15/30) of 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 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 CV-A1, both EV-Cs. The PV-2 detected had VP1 ILE143. Hence, a vaccine strain.The results of this study showed that about 50% of enterovirus infections (including some Sabin PV2s) are being missed by the RD-L20B cell culture based algorithm. This highlights the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-Cs in Nigeria was also confirmed.

Samples showing cytopathology (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.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the

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Introduction. Hepatitis B virus (HBV) infection is the leading cause of hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HBV genotype E (HBV/E) is the predominant genotype in West Africa and has been linked epidemiologically with chronic and occult HBV infections as well as development of HCC. Mutations in the surface and polymerase genes of HBV have been associated with occult infection, drug resistance, vaccine escape, as well as HCC. Hypothesis/Gap Statement. There is limited data on the occurrence and patterns of mutations associated with occult infection, drug resistance, vaccine escape and HCC for HBV/E. Aim. This study characterized amino acid (aa) substitutions in the major hydrophilic (MHR) and reverse transcriptase (RT) regions of the surface and polymerase genes respectively of HBV sequences from a group of Nigerians with genotype E infection. The CpG islands of the PreC/C and PreS/S regions of these sequences were also described. Methodology. HBV surface and polymerase genes were detected using PCR techniques. Occurrence of new and previously described mutations in these genes were analysed using phylogenetic techniques. Results. Overall 13 HBV isolates were each sequenced for polymerase and surface genes mutations. Thirteen and nine PreS/S and PreC/C HBV genes respectively were analysed for CpG islands. Mutations in the MHR and a-determinants region of the S protein were discovered in eleven and nine of the 13 tested isolates respectively. These mutations were concomitant with aa changes in the RT functional domains of the isolates. Mutations associated with vaccine escape, occult infection and poor HCC prognosis were identified in HBV/E isolated in this study. Furthermore, all the isolates had at least one putative nucleotide analogue resistance mutations. Drug resistance mutations had the highest association with CpG islands. Conclusion. The results of this study contribute to further understanding of HBV variability in Nigeria and the West African region. This will aid the planning of adequate HBV immunization and treatment programmes for the countries in the region.

Samples showing cytopathology (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.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were 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 fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.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 (1 isolate).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.

The genomes of seven novel members of previously described DNA and RNA virus families are described here. These viruses were recovered using a viral metagenomic approach from the stool of a drill monkey (Mandrillus leucophaeus) housed in a sanctuary in Cross River State, Nigeria.

The oldest EV-A119 record was in 2008 in a chimpanzee in Cameroon and subsequently in more non-human primates and healthy children. Here we report for the first time the detection of EV-A119 in a child with Acute Flaccid Paralysis, thus suggesting possible association with a clinical condition in humans.