Applied shotgun metagenomics approach for the genetic characterization of dengue viruses

Erley Lizarazo,Natacha Couto,Maria Vincenti-Gonzalez,Erwin C Raangs,Zoraida Velasco,Sarah Bethencourt,Thomas Jaenisch,Alexander W Friedrich,Adriana Tami,John W Rossen

doi:10.1016/j.btecx.2019.100009

Erley Lizarazo, Natacha Couto + Show 8 more

Open Access

https://doi.org/10.1016/j.btecx.2019.100009

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Abstract

Dengue virus (DENV), an arthropod-borne virus, has rapidly spread in recent years. DENV diagnosis is performed through virus serology, isolation or molecular detection, while genotyping is usually done through Sanger sequencing of the envelope gene. This study aimed to optimize the use of shotgun metagenomics and subsequent bioinformatics analysis to detect and type DENV directly from clinical samples without targeted amplification. Additionally, presence of DENV quasispecies (intra-host variation) was revealed by detecting single nucleotide variants. Viral RNA was isolated with or without DNase-I treatment from 17 DENV (1–4) positive blood samples. cDNA libraries were generated using either a combination of the NEBNext® RNA to synthesize cDNA followed by Nextera XT DNA library preparation, or the TruSeq RNA V2 (TS) library preparation kit. Libraries were sequenced using both the MiSeq and NextSeq. Bioinformatic analysis showed complete ORFs for all samples by all approaches, but longer contigs and higher sequencing depths were obtained with the TS kit. No differences were observed between MiSeq and NextSeq sequencing. Detection of multiple DENV serotypes in a single sample was feasible. Finally, results were obtained within three days with associated reagents costs between €130−170/sample. Therefore, shotgun metagenomics is suitable for identification and typing of DENV in a clinical setting.

Highlights

Dengue viruses (DENV) belong to the Flaviviridae family and are among the most widely distributed arthropod-borne viruses worldwide
We evaluated the use of shotgun metagenomics and bioinformatics analyses to detect and type DENV and to reveal the presence of DENV quasispecies directly from sera and plasma samples
From twelve out of seventeen samples, two aliquots were obtained and extracted with and without DNase I treatment to reveal its effect on the number of human and DENV reads

Summary

Introduction

Dengue viruses (DENV) belong to the Flaviviridae family and are among the most widely distributed arthropod-borne viruses worldwide. In the last five decades DENV has rapidly spread around the globe This together with high morbidity rates make DENV a public health threat in tropical and subtropical regions and increasingly in temperate countries [2,3,4]. DENV are single-stranded, positive sense RNA viruses with a genome of approximately 10.7 kb that contain a single open reading frame (ORF) [5]. They comprise 4 antigenically distinct serotypes (DENV-1 to 4) that have up to 65% genome sequence identity [6] and cluster into different genotypes as a result of high mutation rates in their genomes [2,7]. Disease outcome and virus transmission rates have shown to be genotype-dependent [8]

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