Metagenomic sequencing for virus identification in a public-health setting

Abstract

The use of metagenomics for virus discovery in clinical samples has opened new opportunities for understanding the aetiology of unexplained illness. This study explores the potential of this sequence-independent approach in a public-health setting, by systematic analysis of samples cultured from patients with unexplained illness through a combination of PCR-based assays and viral metagenomics. In total, 1834 cell-culture isolates were collected between 1994 and 2007 through the Enterovirus Surveillance programme in the Netherlands. During the 13 year period, seven samples that exhibited reproducible cytopathogenic effects in cell culture tested negative in standard PCR assays for a range of viruses. In order to fill the diagnostic gap, viral metagenomics was applied to these culture supernatants, resulting in the rapid identification of viruses in all of the samples. The unexplained samples contained BK polyomavirus, herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) (which dominated the unexplained samples; n54). The full genomic sequences of four SAFV genotype 3 (SAFV-3) viruses, which share 88-93 % nucleotide identity with known SAFV-3 viruses, are reported. Further screening for SAFV in additional cultured, unidentified clinical isolates from 2008 and 2009 resulted in identification of another SAFV-positive sample. Although the pathogenicity of the identified viruses has not been established, this study demonstrates that viral metagenomics is a powerful tool that can be integrated into public-health monitoring efforts to investigate unidentified viruses in cell cultures from clinical isolates where standard PCR assays fail to detect viruses.

The Fecal Viral Flora of Wild Rodents

“…We describe here the fecal viral flora in several species of wild rodents. Sequences closely and distantly related to known viral sequences were identified as well as many sequences of unknown taxonomic origin [51], [99], [101], [108], [112]–[114]. A fraction of these currently unclassifiable sequences may be derived from still genetically uncharacterized viral families refractory to nucleotide or protein sequence similarity based searches.…”

A Pan-HIV Strategy for Complete Genome Sequencing

“…Cultured virus isolates were used as a model system to demonstrate the HIV-SMART principle and the breadth of diversity it could accommodate. A preponderance of metagenomics papers describing new methods have also relied on cultured isolates or transfected plasmids with lower host backgrounds and higher virus titers (13,(29)(30)(31)(32)(33). However, sequencing directly from plasma is the most expedient workflow and reveals which replication-competent viruses are circulating in the patient.…”

Flaviviruses: Yellow Fever, Japanese B, West Nile, and Others

“…Metagenomics takes this concept further. It is the coordinated study of multiple genomes-the population of genetic material and, as applied to viruses, the entire community of viruses within the host environment [ 34 ]. This approach utilizes direct sequencing and bioinformatics to reconstruct the viral population and as such reveals the genetic diversity and evolution of the virus and its vector hosts.…”

“…Especially powerful is the use of metagenomics when standard techniques fail to identify a viral infection, such as in emerging diseases. For example, metagenomics was used to identify specifi c viruses from samples collected from an enterovirus surveillance program in the Netherlands [ 34 ]. Samples were identifi ed using conventional techniques including PCR that demonstrated cytopathic effects in cell culture without a virus.…”