viernes, 16 de marzo de 2012

METAGENOMICS Y LA IDENTIFICACIÓN DE NUEVOS VIRUS Bexfield N. y Kellam P. 2010

Metagenomics and the molecular identification of novel viruses
Nicholas Bexfield a,⇑, Paul Kellam b
a Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, UK
b The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

a b s t r a c t
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.
2010 Elsevier Ltd. All rights reserved.
Introduction
Given that animal pathogens (in particular viruses) are considered
a significant source of emerging human infections (Cleaveland
et al., 2001), the identification and optimal characterisation of novel
organisms affecting both domestic and wild animal populations
is central to protecting both human and animal health. Recent outbreaks
of human infection caused by influenza H7N7 virus transmitted
from poultry (Koopmans et al., 2004) and H1N1 virus
transmitted from pigs (Dawood et al., 2009; Itoh et al., 2009) are
cases in point, highlighting the need for ongoing, vigilant epidemiological
surveillance of such pathogens in animal populations.
Moreover, epidemiological studies strongly suggest that novel
infectious agents remain to be discovered (Woolhouse et al.,
2008) and may be contributing to a host of cancers, autoimmune
disorders, and degenerative diseases in humans (Relman, 1999;
Dalton-Griffin and Kellam, 2009). Similar, yet-to-be-identified
viruses may be contributing to the pathogenesis of similar diseases
in animals.
Viruses can be identified by a wide range of techniques. Traditional
methods include electron microscopy, cell culture, inoculation
studies and serology (Storch, 2007). Whereas many of the
viruses known today were first identified by these techniques,
the methods have limitations. For instance, many viruses cannot
be cultivated in the laboratory and can only be characterised by
molecular methods (Amann et al., 1995), and in recent years we
have seen the increasing use of these techniques in pathogen discovery
(Fig. 1).
One such approach uses sequence information from known
pathogens to identify related but undiscovered agents through
cross-hybridisation. Examples include microarray (Wang et al.,
2002) and subtractive (Lisitsyn et al., 1993) hybridisation-based
methods. Another advance has involved PCR amplification of the
pathogen genome, where there is complete knowledge of the pathogen
to be amplified (conventional PCR), or where this information
is limited (degenerate PCR). Other PCR methods such as
sequence-independent single primer amplification, degenerate
oligonucleotide primed PCR, random PCR and rolling circle amplification,
also have the capacity to detect completely novel pathogens.
Hybridisation and PCR-based methods are more effective if the
sample to be analysed is first enriched for virus, a process achieved
by removing host and other contaminating nucleic acids. The end
result of most hybridisation and PCR methods are amplified products
that require definitive identification by sequencing. Advances
in sequencing that have facilitated virus discovery include the arrival
of ‘next or second generation sequencing’, which can generate
very large amounts of sequence data.
Technological advances have also resulted in the development
of metagenomics, the culture-independent study of the collective
set of microbial populations (microbiome) in a sample by analysing
the sample’s nucleotide sequence content (Petrosino et al., 2009).
The different microorganisms constituting a microbiome can include
bacteria, fungi (mostly yeasts) and viruses. Examples of
microbiomes in mammalian biology include the microbial populations
inhabiting the human intestine or mucosal surfaces both in
health and disease.
To date, the study of the viral microbiome (virome) has been applied
to a range of biological and environmental samples including
human (Breitbart et al., 2003; Zhang et al., 2006; Finkbeiner et al.,
1090-0233/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2010.10.014

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