Abstract
RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don’t have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.
Highlights
RNA viruses have an enormous impact on human health and constitute a major source of emerging or re-emerging infectious diseases [1], such as those caused by MERS, Ebola, West Nile, Zika, and chikungunya viruses [2,3]
We sequence and de novo assemble the resulting double-stranded RNA (dsRNA) to aid in viral discovery. We refer to this approach as dsRNA-Seq and have used it successfully to identify a variety of RNA viruses in infected animal tissues
By several criteria the two-step method we have developed for purifying dsRNA from total
Summary
RNA viruses have an enormous impact on human health and constitute a major source of emerging or re-emerging infectious diseases [1], such as those caused by MERS, Ebola, West Nile, Zika, and chikungunya viruses [2,3]. High-throughput sequencing of total RNA isolated from infected individuals is a powerful approach to identifying and determining complete genomes of RNA viruses that are potentially responsible for diseases without a known causative agent. This approach allows for the detection of variants of known viruses or synthetic viral agents, which may not be recognized by PCR or serological-based techniques. We have developed an alternative method to enrich for viral RNA sequences that incorporates both negative selection to remove host RNA and positive selection for dsRNA viruses and dsRNA replication intermediates of ssRNA viruses. We refer to this approach as dsRNA-Seq and have used it successfully to identify a variety of RNA viruses in infected animal tissues
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