DETECTION AND IDENTIFICATION OF NON-CULTURABLE ENTEROVIRUSES IN FAECAL SAMPLES FROM ACUTE FLACCID PARALYSIS CASES IN SOUTHWESTERN NIGERIA
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Date
2017
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Abstract
This study revealed the detection and identification of the species of non-culturable enteroviruses present in faecal samples of children with Acute Flaccid Paralysis and characterized the genotype of the enteroviruses by Phylogenetic analysis.Non-culturable enteroviruses are very common, distributed worldwide and go unnoticed and sometimes caused outbreaks in which a larger than usual number of patients develop clinical disease, sometimes with serious consequences.Enteroviruses (EVs) are human pathogens that are very important, associated with various clinical syndromes of which their infections are common in humans across the globe and remain to be a vital public health problem. A total number of sixty (30 cases) culture negative stool suspensions from Southwestern Nigeria were analyzedfor the presence of non-polio Enteroviruses. The study location of six States were included in this study with the children’s age under 15. Viral RNA was extracted from each stool suspension using Total RNA purification kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Eluted Ribonucleic acid (RNA) was used to synthesize complimentary Deoxyribonucleic acid (cDNA) using Script cDNA synthesis kit (Jena Bioscience, Germany) according to the manufacturers’ instructions. Polymerase Chain Reaction (PCR) was carried out using each newly synthesized cDNA. First round PCR was carried out with 35 cycles of amplification and the second round PCR for semi-nested amplification was carried out in three different assays using a constant reverse Primer AN88 and different forward enterovirus species-specific primers AN89, 189 and I87 for Pan-enteroviruses, species A and C Enteroviruses and species B, respectively. Five microliter of the reaction product was separated and visualized on 2 % agarose gel containing 10 ul Ethidium bromide and viewed using a transilluminator. The resulting DNA amplicon was purified and sequenced using the second round primers used for the semi-nested amplification reaction. Generated sequences were manually edited using MEGA 6.06 software. Phylogenetic analysis of the sequenced viruses was done for virus identification by comparing with many references sequences from GenBank. Reference sequences of species A, B and C Enteroviruses was retrieved from GenBank, aligned with generated sequences using the CLUSTAL W programme in MEGA 6.06 software and a maximum Likelihood (ML) tree with 1000 bootstrap replicates constructed using MEGA 6.06 software.
Results showed that of the 30 samples screened, only one was positive showing the expected band size approximately 350bp for enteroviruses VP1 gene. The sample was recovered from a male patient from Oyo State. Only one species (Species B) of enterovirus was recovered from all the samples. Phylogenetic analysis strain showed the sequence as serotype Echovirus 21 (E-21).
In conclusion, this study revealed the prevalence of Enteroviruses (EVs)in south-western Nigeria is low, it furthered generated results about the detection and identification of enterovirus species present in the patient samples which were negative during culturing method. Hence emphasizing the necessity of combination of cultural and molecular method for the detection of non culturable enteroviruses. Also, it documents the first molecular sequence data on Echovirus 21 (E-21) in Oyo State Nigeria and finally characterize the genotype of the enteroviruses by phylogenetic analysis thereby establishing the recovery Echovirus 21 (E-21) of species B in a male child in Oyo State.
Description
xvii, 103p
Keywords
detection and identification, -culturable enteroviruses, Faecal sample, Acute Flaccid paralysis, Phylogenetic analysis