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

Hindawi Publishing Corporation Advances in Virology Volume 2016, Article ID 1412838, 12 pages http://dx.doi.org/10.1155/2016/1412838 Research Article Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay Temitope Oluwasegun Cephas Faleye,1,2 Moses Olubusuyi Adewumi,1 Bamidele Atinuke Coker,3 Felix Yasha Nudamajo,3 and Johnson Adekunle Adeniji1,4 1 Department of Virology, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria Department of Microbiology, Faculty of Science, Ekiti State University, Ado Ekiti, Ekiti, Nigeria 3 Department of Microbiology, Faculty of Science, University of Ibadan, Ibadan, Oyo State, Nigeria 4 WHO National Polio Laboratory, University of Ibadan, Ibadan, Oyo State, Nigeria 2 Correspondence should be addressed to Johnson Adekunle Adeniji; Received 27 December 2015; Accepted 11 February 2016 Academic Editor: George N. Pavlakis Copyright © 2016 Temitope Oluwasegun Cephas Faleye et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. 1. Introduction Enterovirus infections have been associated with an array of clinical manifestations that range from aseptic meningitis through type 1 diabetes to acute flaccid paralysis (AFP) among others [1]. However, these clinically manifest infections represent <10% of the actual burden of enterovirus infections and have been estimated to amount to about 10– 15 million cases annually in the United States alone [2]. The remaining over 90% of such infections are asymptomatic [3]. Enteroviruses are nonenveloped viruses with a diameter of 20–30 nM. Within the virion is a positive sense, single stranded RNA genome that is approximately 7,500 nt long. The genome has one open reading frame (ORF), the polyprotein product of which is autocatalytically cleaved into structural (VP1–VP4) and nonstructural (2A–3D) proteins. The ORF is flanked on both ends by untranslated regions (UTRs) and a poly-A tail at the 3󸀠 -end. Enteroviruses belong to the genus Enterovirus in the family Picornaviridae, order Picornavirales. Classification of enteroviruses used to be based on virion particle structure, tissue culture growth properties, and pathogenesis in humans and animals [4]. However, classification is now based on virus genomics [4] and most especially phylogeny of the VP1 protein [5–16]. Based on the recent classification (http://www .picornaviridae.com/), there are 12 species in the genus, four 2 (Enterovirus species A–species D [EVA-EVD]) of which were previously known as “human enteroviruses.” At the time of writing, EVA contained 25 serotypes made up of 11 CVAs, 10 numbered enteroviruses, and four (4) enteroviruses isolated from nonhuman primates. EVB contained 63 serotypes consisting of one (1) CVA, six (6) CVBs, 28 echoviruses, 27 numbered enteroviruses, and one (1) enterovirus isolated from a nonhuman primate. EVC contained 23 serotypes consisting of nine (9) CVAs, three (3) poliovirus serotypes, and eleven (11) numbered enteroviruses. EVD contained five (5) serotypes consisting only of numbered enteroviruses (http:// www.picornaviridae.com/). Besides the fact that EVB has the highest number of serotypes, it is also the most commonly detected [15–21]. It has however been suggested that this phenomenon (called the EVB bias) might be an artefact of the strategy used for enterovirus isolation and might not be truly representative of the enterovirus diversity landscape [21, 22]. Almost all previous studies documenting enterovirus diversity in Nigeria [15, 16, 23, 24] clearly showed the preponderance of EVB. However, all such studies have been cellculture based and mainly used the RD cell line which has been suggested to be the EVB bias [15–21], for enterovirus isolation. The only study that did differently [22] used MCF 7 and LLCMK2 cell lines for enterovirus isolation and documented an increase in the detection rate of enterovirus species C (EVC) members. Recently, Nix et al.’s [25] cell-culture independent protocol for direct detection of enteroviruses from clinical specimen was recommended [4] for enterovirus surveillance alongside the previously established protocols [26, 27]. In this study, we investigated whether this strategy will show the same enterovirus diversity landscape as the established cellculture dependent protocols [26, 27] and document a preponderance of EVAs in Southwestern Nigeria. 2. Methodology 2.1. Sample Collection and Storage. Faecal samples were collected from sixty (male = 37, female = 23) apparently healthy children aged 1 to 10 years attending public primary schools in Ibadan, Nigeria. Samples were collected from the pupils after approval and consent were secured from the school administration and the guardian or parents of the children, respectively. Stool samples were collected from each of the children into appropriately labelled sterile collection bottles. Samples were then transported to the laboratory in the Department of Virology, College of Medicine, University College Hospital, Ibadan, Nigeria, in a cooler filled with ice packs to maintain a temperature of about 4∘ C. On arrival at the laboratory, the stool specimens were stored at −20∘ C until analysis. 2.2. Sample Processing. About one gram of each stool specimen was diluted in 3 mL phosphate buffered saline (PBS), 1 mL chloroform, and one gram of glass beads. The mixture wa (...truncated)