Unbiased, Metatranscriptome-based Detection and Phylogeny of Alphavirus Directly from Culex cedecei Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol

The trajectory of molecular biology hardware development in recent years has trended towards a hand-held, ultra-portable form factor. Products such as the two3™ qPCR platform from Biomeme Inc., as well as the MinION™ from... [ view full abstract ]

The trajectory of molecular biology hardware development in recent years has trended towards a hand-held, ultra-portable form factor. Products such as the two3™ qPCR platform from Biomeme Inc., as well as the MinION™ from Oxford Nanopore Technologies, have generated renewed prospects for point-of-care clinical diagnostics and point-of-sampling, near-real-time environmental biosurveillance of viral and microbial pathogens. Despite continual improvement of throughput and quality of nanopore sequencing reads, questions on the practical utility of this technology remain. Demonstration of successful use of these tools, particularly nanopore sequencing, from diverse, complex sample types (i.e., non-sterile, non-clinical) is required to accelerate the full realization of this technology. Here, using ultra-portable molecular biology hardware, samples of Culex cedecei mosquitoes collected in the Florida Everglades ecosystem were screened for Venezuelan Equine Encephalitis Virus (VEEV), an encephalitic RNA-virus. A single 20-mosquito sample pool was found positive for VEEV by real-time reverse transcription qPCR on the Biomeme two3. The positive sample was strain-typed using nanopore sequencing of the metatranscriptome, and determined to contain Everglades Virus (EVEV), a strain of VEEV transmitted solely by Culex cedecei in South Florida. The result was later confirmed on ‘gold standard’ PCR systems and by sequencing on an Illumina MiSeq. This work includes the first demonstration of unbiased, metatranscriptome-based, strain-level detection of a pathogen from a complex environmental sample matrix using nanopore sequencing.