Abstract
BackgroundThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. AimTo investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples.MethodsWe performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study.ResultsDirect metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. ConclusionsThis work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.
Highlights
Arboviruses are predominantly RNA viruses that replicate in haematophagous arthropod vectors such as ticks, mosquitoes and other biting flies to maintain their transmission cycle [1]
Human disease outbreaks caused by arboviruses have increased in prevalence since the 2000’s, led by the spread of mosquito-borne arboviruses such as chikungunya (CHIKV), dengue (DENV), West Nile (WNV), yellow fever (YFV) and Zika (ZIKV) viruses across both hemispheres [2]
11,082/11,826 due to a higher proportion, 744/11826, of ambiguous base calls, suggesting 1D reads are most suitable for this approach. These results clearly show that there are considerable levels of viral nucleic acid present in a large proportion of CHIKV and dengue virus (DENV) qRT-PCR positive clinical samples, and demonstrate that relatively modest metagenomic sequencing is capable of elucidating significant portions of viral genome even for samples with cycle threshold (Ct) values at the higher end of clinical range
Summary
Arboviruses are predominantly RNA viruses that replicate in haematophagous (blood-sucking) arthropod vectors such as ticks, mosquitoes and other biting flies to maintain their transmission cycle [1]. CHIKV is a single-stranded positive-sense RNA virus of the alphavirus genus, which causes the debilitating arthritic disease, chikungunya [4] It has spread globally and been designated a serious emerging disease by the World Health Organization [5]. Conclusions: This work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. It explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION
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