Viral metagenomics

Abstract

Characterisation of new viruses is often hindered by difficulties in amplifying them in cell culture, limited antigenic/serological cross-reactivity or the lack of nucleic acid hybridisation to known viral sequences. Numerous molecular methods have been used to genetically characterise new viruses without prior in vitro replication or the use of virus-specific reagents. In the recent metagenomic studies viral particles from uncultured environmental and clinical samples have been purified and their nucleic acids randomly amplified prior to subcloning and sequencing. Already known and novel viruses were then identified by comparing their translated sequence to those of viral proteins in public sequence databases. Metagenomic approaches to viral characterisation have been applied to seawater, near shore sediments, faeces, serum, plasma and respiratory secretions and have broadened the range of known viral diversity. Selection of samples with high viral loads, purification of viral particles, removal of cellular nucleic acids, efficient sequence-independent amplification of viral RNA and DNA, recognisable sequence similarities to known viral sequences and deep sampling of the nucleic acid populations through large scale sequencing can all improve the yield of new viruses. This review lists some of the animal viruses recently identified using sequence-independent methods, current laboratory and bioinformatics methods, together with their limitations and potential improvements. Viral metagenomic approaches provide novel opportunities to generate an unbiased characterisation of the viral populations in various organisms and environments.

Viral metagenomics as an emerging and powerful tool in veterinary medicine

“…characterization (Ambrose and Clewley 2006;Delwart 2007;Bexfield and Kellam 2010). Some of the more commonly used sequence-independent strategies are briefly described in the following sections as well as a number of different sequencing technologies.…”

“…To find novel viruses, other tools have to be developed. Toward this end, many researchers are investigating if it is possible to use motifs, such as diand tri-nucleotide frequencies, codon usage, and secondary structures, to distinguish viral sequences (Delwart 2007;Trifonov and Rabadan 2010). …”

“…This procedure aims to provide the genetic composition of the complete viral populations of a sample in an unbiased and culture-independent manner (Handelsman 2004;Delwart 2007). This approach has been successfully used to investigate viral populations in different environments such as seawater (Breitbart et al 2002), gastrointestinal tracts (Victoria et al 2009), and respiratory samples (Allander et al 2007) and have demonstrated that there is a high diversity among viruses, and that there is a vast number of viruses that are yet to be discovered.…”

“…The use of hybridization-based approaches, such as microarray and subtractive hybridization, can be found in other reviews (Ambrose and Clewley 2006;Delwart 2007;Bexfield and Kellam 2010;Tang and Chiu 2010). It also discusses shortly how to follow up on the findings and what the different possible viral metagenomic applications in veterinary science are.…”

“…Thus, one of the most important, yet difficult, tasks of viral metagenomics is to prepare the sample in such a way that the contaminating host nucleic acids are depleted while the viral nucleic acids are preserved (Delwart 2007). Different strategies, which are usually based on the special properties of viruses, have been used for this purpose.…”