Abstract
The future of infectious disease surveillance and outbreak response is trending towards smaller hand-held solutions for point-of-need pathogen detection. Here, samples of Culex cedecei mosquitoes collected in Southern Florida, USA were tested for Venezuelan Equine Encephalitis Virus (VEEV), a previously-weaponized arthropod-borne RNA-virus capable of causing acute and fatal encephalitis in animal and human hosts. A single 20-mosquito pool tested positive for VEEV by quantitative reverse transcription polymerase chain reaction (RT-qPCR) on the Biomeme two3. The virus-positive sample was subjected to unbiased metatranscriptome sequencing on the Oxford Nanopore MinION and shown to contain Everglades Virus (EVEV), an alphavirus in the VEEV serocomplex. Our results demonstrate, for the first time, the use of unbiased sequence-based detection and subtyping of a high-consequence biothreat pathogen directly from an environmental sample using field-forward protocols. The development and validation of methods designed for field-based diagnostic metagenomics and pathogen discovery, such as those suitable for use in mobile “pocket laboratories”, will address a growing demand for public health teams to carry out their mission where it is most urgent: at the point-of-need.
Highlights
With increasing accessibility of metagenomics- and metatranscriptomics-based analyses, clinicians and researchers have begun to embrace the technology as a means of detection for unknown etiological agents of disease[1,2,3,4,5,6,7,8,9]
During the Zika Virus (ZIKV) outbreak of 2015–2016 in Brazil, several groups used nanopore sequencing of RT-qPCR amplicons from mosquito samples to track incidence of ZIKV infection and study ZIKV vector dynamics[37,38]
This is likely due to the lower sequencing depth of nanopore data relative to second-generation sequencing machines, and subsequent detection of predominantly host genomic material. Overcoming this challenge will enable genome-based biosurveillance without the constraint of PCR primer design and optimization. This would be useful for monitoring arbovirus and other viral hemorrhagic fever (VHF) vectors in hot-spot regions throughout the world where frequent epizootic events threaten the health of human populations
Summary
With increasing accessibility of metagenomics- and metatranscriptomics-based analyses (meta-omics), clinicians and researchers have begun to embrace the technology as a means of detection for unknown etiological agents of disease[1,2,3,4,5,6,7,8,9]. To date, there have been no reports of unbiased (non-PCR) strain-level detection of specific organisms-of-interest directly from environmental sample matrices (e.g., non-clinical, non-sterile, non-laboratory derived) using nanopore sequencing. This is likely due to the lower sequencing depth of nanopore data relative to second-generation sequencing machines, and subsequent detection of predominantly host genomic material. Overcoming this challenge will enable genome-based biosurveillance without the constraint of PCR primer design and optimization. This work demonstrates the practical utility of hand-held thermocyclers and nanopore sequencing devices for unbiased strain-level detection of arboviruses from complex, environmental sample matrices suitable for use in field-based “pocket laboratories”[48]
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