Metagenomics and the molecular identification of novel viruses

Abstract

There have been rapid recent developments in establishing methods for identifying and characterising viruses associated with animal and human diseases. These methodologies, commonly based on hybridisation or PCR techniques, are combined with advanced sequencing techniques termed 'next generation sequencing'. Allied advances in data analysis, including the use of computational transcriptome subtraction, have also impacted the field of viral pathogen discovery. This review details these molecular detection techniques, discusses their application in viral discovery, and provides an overview of some of the novel viruses discovered. The problems encountered in attributing disease causality to a newly identified virus are also considered.

Fig. 1

A schematic overview of the molecular methods currently available for viral discovery. Hybridisation methods include microarray and subtractive hybridisation techniques such as representational difference analysis. PCR-based methods include degenerate PCR, degenerate oligonucleotide primed PCR (DOP-PCR), sequence-independent single primer amplification (SISPA), random PCR and rolling circle amplification (RCA).

Fig. 2

Sequence of events in the molecular detection of viruses: (A) Samples processed by hybridisation or PCR require steps to enrich for virus before amplified products are sequenced and identified. Enrichment may result in decreased assay sensitivity, and amplification can generate bias towards a dominant sequence; (B) Transcriptome subtraction methods can be performed without enrichment or amplification with direct sequencing of nucleic acids extracted from a sample of interest. Subsequent subtraction of resulting sequences from databases facilitates virus identification.

Fig. 3

Graphic representation of the probability of detecting viral sequences based on the viral genome-transcript sequence frequency and the number of sequence ‘reads’ generated (lines with symbols).