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Phylogenic Analysis of Human Bocavirus Detected in Children With View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Archive Ouverte en Sciences de l'Information et de la Communication Phylogenic analysis of human bocavirus detected in children with acute respiratory infection in Yaounde, Cameroon Sebastien Kenmoe, Marie-Astrid Vernet, Mohamadou Njankouo-Ripa, Véronique Beng Penlap, Astrid Vabret, Richard Njouom To cite this version: Sebastien Kenmoe, Marie-Astrid Vernet, Mohamadou Njankouo-Ripa, Véronique Beng Penlap, Astrid Vabret, et al.. Phylogenic analysis of human bocavirus detected in children with acute respira- tory infection in Yaounde, Cameroon. BMC Research Notes, BioMed Central, 2017, 10, pp.293. 10.1186/s13104-017-2620-y. hal-02366923 HAL Id: hal-02366923 https://hal-normandie-univ.archives-ouvertes.fr/hal-02366923 Submitted on 19 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Kenmoe et al. BMC Res Notes (2017) 10:293 DOI 10.1186/s13104-017-2620-y BMC Research Notes RESEARCH NOTE Open Access Phylogenic analysis of human bocavirus detected in children with acute respiratory infection in Yaounde, Cameroon Sebastien Kenmoe1,2,3, Marie‑Astrid Vernet1, Mohamadou Njankouo‑Ripa1, Véronique Beng Penlap2, Astrid Vabret3 and Richard Njouom1* Abstract Objective: Human Bocavirus (HBoV) was frst identifed in 2005 and has been shown to be a common cause of respiratory infections and gastroenteritis in children. In a recent study, we found that 10.7% of children with acute respiratory infections (ARI) were infected by HBoV. Genetic characterization of this virus remains unknown in Central Africa, particularly in Cameroon Leeding us to evaluate the molecular characteristics of HBoV strains in Cameroonian children with ARI. Results: Phylogenetic analysis of partial HBoV VP1/2 sequences showed a low level of nucleotide variation and the circulation of HBoV genotype 1 (HBoV-1) only. Three clades were obtained, two clustering with each of the reference strains ST1 and ST2, and a third group consisting of only Cameroon strains. By comparing with the Swedish refer‑ ence sequences, ST1 and ST2, Cameroon sequences showed nucleotide and amino acid similarities of respectively 97.36–100% and 98.35–100%. These results could help improve strategies for monitoring and control of respiratory infections in Cameroon. Keywords: Human bocavirus, Children, Acute respiratory infection, Cameroon, Africa Introduction studies have observed viral particles having the char- During the past two decades, new molecular techniques acteristics of this family of viruses; small DNA viruses have contributed to the discovery of many viruses. Tis with unenveloped icosahedral capsid and a diameter was the case in September 2005 with the discovery of the ranging from 18 to 26 nm. Te HBoV genome is a 5.2 kb Human Bocavirus (HBoV) by a Swedish research team linear negative sense single-stranded DNA. During the [1]. HBoV was subsequently named HBoV-1. Since, three 8th ICTV report it was decided that the diferent spe- other species (HBoV-2, 3, and 4) have been described cies of the genus Bocavirus should show a similarity in from stool samples in Pakistan, Australia, and Nigeria the NS1 gene of at least 95%. Based on the analysis of a respectively [2–4]. Based on phylogenetic analysis of large number of VP1 gene sequences and calculations HBoV genomic sequences, it was assigned to the Par- of the genetic distance of complete genomes of difer- voviridae family, Parvovirinae sub-family, and Bocavi- ent species of HBoV, Kapoor and collaborators recently rus genus. Indeed, the sequences identifed were closely proposed a new classifcation mode for HBoV suggest- related to two other bocaviruses: the Bovine Parvovirus ing that HBoV strains with more than 8 and 10% difer- and the canine Minute Virus. Although this classifca- ence respectively in protein and nucleotide sequences tion was based on genetic characterization, microscopy of the VP1 complete gene should be considered as dif- ferent species. Tose with more than respectively 1.5 *Correspondence: njouom@pasteur‑yaounde.org and 5% diference should be considered as diferent 1 Virology Unit, Centre Pasteur of Cameroon, Yaounde, BP 1274 Yaounde, genotypes [3]. Tis proposal is consistent with the cur- Cameroon Full list of author information is available at the end of the article rent nomenclature of the four HBoV species. A decade © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kenmoe et al. BMC Res Notes (2017) 10:293 Page 2 of 5 after the identifcation of HBoV-1 in Sweden, it has been semi-nested reaction with 2 µL of the frst PCR product detected at frequencies ranging from 2 to 19% [5] in sev- was performed with the pair of primer VB-B and VP-C eral countries across Europe [6–9], Asia [10–13], Amer- (5′-CTTAGAACTGGTGAGAGCACTG-3′) according to ica [14–17], Africa [18–21], Middle East [22–24], and the previous protocol. Te cycling parameters for the two Australia [4, 25], revealing an overall distribution of this PCR included an enzyme activation and initial denatura- virus. Tese studies indicate that HBoV-1 is primarily tion at 94 °C (3 min); 40 cycles of denaturation at 94 °C a respiratory pathogen. HBoVs are detected not only in (45 s), primer annealing at 50 °C (30 s) and extension at the respiratory tract (HBoV-1 and 2), but also in the gas- 72 °C (1 min 30 s); a fnal extension at 72 °C (10 min), trointestinal tract (HBoV-1 to 4) where they are associ- and a hold at 4 °C. Te amplifed VP1/2 capsid of 980 bp ated with gastroenteritis [5]. HBoV are highly conserved product was observed after electrophoresis in a 1.5% aga- viruses and sequences obtained in various regions of the rose gel. world by several groups highlight a high homology of Te VP1/2 capsid PCR products were purifed and genetic sequences [7, 9, 11, 18, 22]. sequenced using Big Dye Terminator cycle sequenc- In a recent study, we have found that 10.7% of children ing (Applied Biosystems, Foster City, USA, Cat No. with acute respiratory infections (ARI) were infected by a 4,337,454) and semi-nested PCR primers according HBoV [21]. However, most molecular epidemiology stud- manufacturer instructions. Electrophoresis and data col- ies of HBoVs have been performed in developed coun- lection were done on an Applied Biosystems AB3100 tries [5]. Genetic characterization of this virus remains genetic analyzer. Sequences were frst aligned and edited unknown in most African countries, particularly in Cam- using Sequence Navigator1 software (Applied Biosys- eroon. Tis study is, to our knowledge, the frst analysis of tems, Foster City, CA). Te consensus sequence was gen- the genetic characteristics of HBoVs obtained from Cam- erated by removing low quality base peaks at the end of eroonian children with acute respiratory infections (ARI). the chromatogram and correcting base pair mismatches. Methods Phylogenetic analysis Study population and samples Genotypes were determined by phylogenetic analysis Te study population was hospitalized and outpatient comparing the consensus sequence of each sample to ref- children with ARI consulting at the pediatric service erence VP1/2 capsid sequences including the Swedish ref- of ‘Centre Hospitalier d’Essos’ in Yaounde, Cameroon. erence sequences ST1 and ST2, and reference sequences Demographic and clinical characteristics of these chil- of HBoV-2 (NC_012042), HBoV-3 (NC_012564), dren have been previously described [21]. For this study HBoV-4 (NC_012729), BPV (NC_001540.1), and MVC nasopharyngeal swab samples were used. (NC_004442.1). Phylogenetic analyses were performed using MEGA Laboratory analysis software version 6 [26]. Briefy, sequences in FASTA for- DNA was extracted using QIAamp DNA Mini kit (Qia- mat were aligned using Clustal W algorithm. Genetic gen, Hiden, Germany, Cat No. 51306) from nasopharyn- distances were measured with the Kimura-2 parameter geal swab specimens following the manufacturer’s model. A phylogenetic tree was constructed with the instructions. A fnal elution volume of 200 µL of DNA neighbor-joining algorithm and robustness of the tree was stored a −80 °C prior to testing by a commercially was evaluated with 1000 bootstrap resamplings. available duplex real time PCR for the detection of human Te MEGA software version 6 was used to generate Adenovirus and HBoV (Respiratory Multi Well System predicted amino acid sequences from consensus nucleo- r-gene™, BioMerieux, Lyon, France, Cat No. 1092480) tide sequences by translation. Te comparison of the sites following the manufacturer’s instructions. HBoV posi- and proportion of similarities at amino acid and nucle- tive samples were then selected and a fragment of the otide levels was made between consensus amino acid VP1/2 capsid gene was amplifed by semi-nested PCR as and nucleotide sequences of each Cameroonian HBoV described elsewhere [18]. Briefy, 10 µL of the DNA was sequences and ST1 and ST2 reference sequences.
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