Available online at www.sciencedirect.
com
Exploring the viral world through metagenomics
Karyna Rosario and Mya Breitbart
Viral metagenomics, or shotgun sequencing of purified viral
particles, has revolutionized the field of environmental virology
by allowing the exploration of viral communities in a variety of
sample types throughout the biosphere. The introduction of
viral metagenomics has demonstrated that dominant viruses in
environmental communities are not well-represented by the
cultured viruses in existing sequence databases. Viral
metagenomic studies have provided insights into viral ecology
by elucidating the genetic potential, community structure, and
biogeography of environmental viruses. In addition, viral
metagenomics has expanded current knowledge of virus–host
interactions by uncovering genes that may allow viruses to
manipulate their hosts in unexpected ways. The intrinsic
potential for virus discovery through viral metagenomics can
help advance a wide array of disciplines including evolutionary
biology, pathogen surveillance, and biotechnology.
Address
University of South Florida, College of Marine Science, 140 7th Avenue
South, Saint Petersburg, FL 33701, United States
Corresponding author: Breitbart, Mya (mya@marine.usf.edu)
Current Opinion in Virology 2011, 1:289–297
This review comes from a themed issue on
Viral genomics
Edited by Elodie Ghedin and Christipher Upton
Available online 2nd July 2011
1879-6257/$ – see front matter
# 2011 Elsevier B.V. All rights reserved.
DOI 10.1016/j.coviro.2011.06.004
Introduction
The process of viral particle purification followed by shot-
gun sequencing, known as viral metagenomics, has
enabled exploration of the genetic diversity contained
within the most abundant biological entities in the bio-
sphere. Viral metagenomics circumvents limitations
associated with traditional virus characterization methods,
such as PCR or microarrays, and the power of this method
resides in targeting total viral nucleic acids without the
need for ‘a priori’ knowledge of the viral types present
[1,2]. In contrast to standard metagenomic approaches that
sequence total DNA [3,4], the initial viral particle purifi-
cation step in viral metagenomic projects ensures that most
of the sequencing effort results in the detection of viral
nucleic acids. This concept was first applied to discover
viruses in serum samples from patients with diseases of
unknown etiology by amplifying viral nucleic acids that
www.sciencedirect.com
had been digested with restriction enzymes before ampli-
fication and sequencing [5]. The first study investigating
viral communities in an environmental sample through
metagenomics improved these methods by mechanically
shearing DNA to allow the random amplification of total
viral nucleic acids regardless of restriction sites [6]. The
introduction of next generation sequencing technologies
has significantly increased the throughput of viral meta-
genomics in the past five years [7]. Almost a decade after
the first environmental viral metagenome metagenome
was sequenced, viral metagenomics has proven to be an
indispensable tool for understanding viral community
structure and discovering novel genes and viruses
(Figure 1).
Metagenomics ‘dark matter’: the vast
unknown
To date there are 24 published studies investigating viral
communities in environmental samples through viral
metagenomics (Table 1). It is clear that environmental
viral communities are different from well-characterized
viruses as studies consistently report more than 50% of
the sequences as unknown. Environmental viral metage-
nomic studies have produced more than 810 Mbp of data
for which there are no similarities in GenBank. These
sequences, representing about 70% of the data that has
been generated through environmental viral metage-
nomics, remain in an ever-growing pool of unknowns.
Further analysis of this unknown pool is complicated by
the lack of a centralized database for submitting and/or
retrieving viral metagenomic data (Table 1). Moreover,
many of the ‘known’ viral sequences in environmental
viromes have low amino acid similarities (<50%) to
known viral proteins and, thus, represent undescribed
viral species [8,9]. The vast novelty of this genetic infor-
mation has brought attention to viruses, especially phages
(i.e. viruses that infect prokaryotes) [7,10], as the largest
reservoir of untapped genetic information in the bio-
sphere.
The high percentage of unknown sequences in environ-
mental viromes is consistent with findings from cultured
virus genomes. Approximately 30% of open reading
frames (ORFs) in sequenced viral genomes are ORFans,
which have no homologs in the known prokaryotic or viral
world, compared to only 9% ORFans in bacterial genomes
[11]. These observations are further supported by the
high percentage of unknown sequences in environmental
viromes (>50%) compared to microbial metagenomes
(10%) [1], suggesting that viral diversity is significantly
larger than that among bacteria. These findings highlight
our lack of understanding of viral proteins and stress the
Current Opinion in Virology 2011, 1:289–297
290 Viral genomics

Figure 1

INDUSTRY
[Functional Viral Metagenomics]

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kn

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Un

nz
lE
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No
ota
Ann

thogens
Emerging Pa

DISCOVERY SURVEILLANCE
[Vector-Enabled Metagenomics]
New Viral Species

EVOLUTIONARY
GAPS

VIRAL
NUCLEIC ACIDS
er
ransf
ions

ne T
ract

Ge
Inte

ta l
on
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os

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ECOLOGY
Current Opinion in Virology

Applications of viral metagenomics. Tapping into environmental viral nucleic acids has allowed the scientific community to gain a better understanding
of viral ecology (i.e. viral diversity, community structure, and biogeography); discover novel viral species that may help elucidate evolutionary gaps;
investigate virus–host interactions (i.e. antiviral responses and lysogeny); identify auxiliary metabolic genes obtained through horizontal gene transfer
that may affect host functionality; design virus surveillance strategies for emerging pathogens; and discover viral enzymes with commercial and/or
biomedical value.

need for biochemical data which overwhelmingly lag
behind genomics in this sequencing era.
Despite the inability to identify most of the sequences
produced through viral metagenomics, studies have taken
advantage of this technique to catalogue viruses in environ-
mental samples based on the identifiable sequences and
investigate community composition through statistical
analyses (see [12–14] for an overview of metagenomic data
analysis). The complete genomes of novel viruses have
been assembled from environmental viromes [15–17,18],
and mathematical modeling based on viral metagenomic
sequences has yielded insight into community structure
and biogeography [6,7]. In addition, several studies are now
starting to link metagenomic data from viruses with
potential hosts based on genomic signatures and phylo-
genetic analyses [17,19–21]. Through these routes, meta-
genomic studies have re-fueled the field of viral ecology,
providing unprecedented views into viral diversity and
virus–host interactions, and leading to the discovery of
novel viruses.
Metagenomics and viral ecology
Data generated through viral metagenomic studies have
been used to tackle questions, such as viral biogeography,
that could not be addressed with traditional methods.
Amplification of signature genes for specific viral groups
suggested that certain viruses are found globally in

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Table 1

Published environmental viral metagenomes

Sample type a Location Sequencing b Target Database c Accession number

Coastal seawater [6]
Coastal sediment [54]
Open ocean and
coastal waters [7]
Coastal seawater [55]
Estuary [56]
Soil [25]
Aquaculture system and
solar salterns [57]
Saltern [58]
Rice paddy soil [59]
Hot springs [23]
Marine and freshwater
microbialites [26]
EBPR sludge [30]
Estuary [34]
Coral (P. compressa) [60]
California S dsDNA GSS AY079522.1–AY079585.1AY080585.1 (SP)
BH898061.1–BH898933.1 (MB)
California S dsDNA GSS CC821301.1–CC822456.1
Sargasso Sea, British Columbia, P ssDNA, dsDNA CAMERA CAM_PROJ_MarineVirome
Gulf of Mexico, and Arctic
Canada S RNA GSS DX421099.1–DX421142.1 (SOG)
DX420985.1–DX421098.1 (JP)
Chesapeake Bay S dsDNA CAMERA CAM_PROJ_CBVIRIO
Peru (Rainforest), California S dsDNA GSS ER781257–ER785833
(Desert), Kansas (Prairie)
Salton Sea and Chula Vista, P ssDNA, dsDNA SEED See footnote d
California
Alicante, Spain S dsDNA GenBank DQ238866 (EHP-1); EF503711–EF503714 (EHP-2; EHP-3)
Daejeon, Korea S ssDNA, dsDNA GenBank ABQX01000001–ABQX01000878
Yellowstone, USA S dsDNA CAMERA CAM_PROJ_ViralSpring
Bahamas and Mexico P ssDNA, dsDNA SEED 4440323.3 (Highborne Cay)
4440320.3 (PozasAzules)
4440321.3 (Rio Mesquites)
Madison, Wisconsin S dsDNA IMG/M Taxon ID: 2007300000
Tampa Bay, Florida P ssDNA, dsDNA SEED 4440102.3
Kane’ohe Bay, Hawaii P ssDNA, dsDNA SEED 4440376.3 (t = 0); 4440374.3 (control); 440370.3 (DOC);
440371.3 (pH); 44403777.3 (nutrient); 4440375.3 (temperature)

Environmental viral metagenomics Rosario and Breitbart 291

Coral (D. strigosa) [61]
Hydrothermal vents [62]
Reclaimed and
potable water [9]
Man-made lake [8]
Antarctic lake [63]
Current Opinion in Virology 2011, 1:289–297
Mount Irvine Bay in S dsDNA WGS ABVU01000001–ABVU01001580 (DsH)
Buccoo, Tobago ABVT01000001–ABVT01000930 (DsB)
East Pacific Rise S dsDNA GSS ED017434–ED017695 (RAPD)
ED017696–ED017873 (LASL)
Manatee County, Florida P ssDNA, dsDNA, RNA CAMERA CAM_PROJ_ReclaimedWaterViruses
Maryland S, P RNA ftp site ftp://ftp.jcvi.org/pub/data/lakemeta
Lake Limnopolar P ssDNA, dsDNA SEED 4441778.3 (Spring); 4441558.3 (Summer)

Saltern [21] Alicante, Spain S dsDNA GenBank GU735099–GU735367; GU735369–GU735406;

HM030731–HM030733
Fermented food [64] Korea P dsDNA SRA SRP002583
Activated sludge [65] Auburn, Alabama S dsDNA TA 2251203077–2251204802
Hypersaline lake [24] Senegal S ssDNA, dsDNA GSS GS926706–GS927688
Hydrothermal vents [28] Juan de Fuca Ridge P dsDNA CAMERA CAM_SMPL_A0003
a
References for each metagenomic study are listed with the sample type.
b
Refers to the sequencing technology used, that is, Sanger (S) or Pyrosequencing (P).
c
Indicates the database where metagenomic sequences can be found including GenBank’s Genome Survey Sequence (GSS), Whole Genome Sequences (WGS), Short Read Archive (SRA), and
Trace Archive (TA), as well as platforms dedicated to analyze metagenomic datasets including CAMERA (http://camera.calit2.net/), SEED (ftp site: ftp://ftp.theseed.org), and Integrated Microbial
Genomes (IMG/M: http://img.jgi.doe.gov/cgi-bin/m/main.cgi).
d
The following libraries can be downloaded from SEED: 4440436.3 (low salinity; D–V), 4440432.3 (low salinity; E–V), 4440420.3 (low salinity; F–V), 4440427.3 (medium salinity; K–V), 4440428.3
(medium salinity; I–V), 4440431.3 (medium salinity; G–V), 4440417.3 (medium salinity; J–V), 4440421.3 (high salinity; L–V), 4440145.4 (high salinity; N–V), 4440144.4 (high salinity; M–V), 4440414.3
(freshwater; B2–V), and 4440424.3 (freshwater; A–V).
292 Viral genomics
extremely different environments supporting the hypoth-
esis that ‘everything is everywhere’ [22]. Metagenomics
opened the opportunity to test this hypothesis using
genetic information from the viral community as a whole
rather than using specific, well-known viral groups. Stat-
istical comparisons between different marine DNA vir-
omes suggested that the vast majority of viral types are
globally distributed in seawater but the relative abun-
dance of viral types differs between locations [7]. Viral
metagenomics has also shown that specific viral
sequences are locally and globally conserved in hot
springs with different physical and biogeochemical prop-
erties [23] as well as in geographically distant hypersaline
environments [24]. However, this global distribution may
not hold true for all environments. Comparisons between
soil viromes indicated that the viral communities in
different soil types are genetically unique with little or
no phylogenetic overlap [25]. Likewise, metagenomic
data indicated that freshwater and marine microbialites
from different locations have distinct viral communities
[26]. Initial data suggest that viral biogeography depends
on the environmental matrix; however, deeper sequen-
cing may reveal global distribution of viruses.
Metagenomics can also provide insight into virus–host
interactions, which is critical for understanding the co-
evolution of host and viral genomes. The clustered reg-
ularly interspaced short palindromic repeat (CRISPR)
system is an antiviral mechanism found in archaea and
bacteria in which genomic sequences from predatory
viruses are integrated as CRISPR spacers in the host
genome, rendering the host ‘immune’ to these viruses
[27]. CRISPR spacer sequences therefore provide a direct
link between viruses and their uncultivated prokaryotic
hosts. Studies have taken advantage of this CRISPR
feature to elucidate potential microbial hosts of viral
assemblages [28,29] and determine if a given population
has been previously infected by specific phages [30].
Comparison of environmental viral communities with
their respective microbial host populations through meta-
genomics has suggested that silent mutations and exten-
sive recombination in viral genomes may help viruses
evade the CRISPR system [29,31].
Another important virus–host interaction explored
through metagenomics is the stable symbiotic state
known as lysogeny. Lysogeny also involves the integ-
ration of viral sequences into the prokaryotic host gen-
ome, but in this case the whole viral genome is integrated.
This association may be beneficial to both host and virus
and is thought to be more prevalent in oligotrophic and/or
extreme environments [32]. In hydrothermal vents, meta-
genomic studies suggest that temperate phages (phages
with the ability to integrate into host genomes) within a
vent site are a less diverse subset of the virioplankton
community [33]. Data gathered from temperate marine
phage metagenomes have been used to design quantitative
Current Opinion in Virology 2011, 1:289–297
assays to examine the expression levels of specific lyso-
geny-related genes [34].
Cellular genes in viral metagenomes
Sequences similar to metabolic genes from cellular organ-
isms are consistently recovered in environmental viromes.
This finding is unexpected since viral particles are pur-
ified before processing, and initially raised concerns
regarding possible host contamination in the viromes.
These metabolic genes may also represent gene transfer
agents (i.e. virus-like particles involved in bacterial gene
transfer that package random fragments of host DNA)
[35,36]. However, metabolic genes have been detected in
the genomes of completely cultured phages and eukar-
yotic viruses, where they are known as auxiliary metabolic
genes (AMGs) [37]. By investigating the function and
genomic context of AMGs, bioinformatic studies have
documented that there is little restriction to the types of
AMGs carried by environmental viruses [10,38,39,40].
Data-mining studies are constantly expanding the suite of
known viral AMGs and suggest that the acquisition of
metabolic genes by viruses is not random, with viromes
enriched in AMGs that are beneficial for survival in a
given environment [33,39]. Metagenomic data have
been used to design quantitative assays to detect envir-
onmentally relevant AMGs, such as genes related to
photosynthesis and nutrient stress, revealing that some
of these genes can be found on the order of 106 copies per
liter in the marine environment [33].
Most studies of AMGs have focused on genes that are
relevant for oceanic nutrient and biogeochemical cycles.
However, viromes contain a diversity of AMGs, such as
those involved in motility and chemotaxis, antioxidation,
transcriptional repression, translation and post-translation
modification, which suggests that viruses manipulate
their hosts in unforeseen ways [39,40,41]. Recent dis-
covery of a phage gene homologous to peptide deformy-
lases and other genes involved in translation suggests that
phages may control both phage and host protein expres-
sion at the translational level [39]. Furthermore, viral
metagenomics resulted in the discovery of a phage-
encoded transcriptional repressor that can potentially
help the phage evade host antiviral mechanisms [18].
The diversity and widespread distribution of AMGs in
different environmental viromes provide further evi-
dence of rampant horizontal gene transfer between hosts
and viruses [42,43] by shedding light on genes not com-
monly associated with the viral gene pool that may shape
the evolution and functionality of both prokaryotic and
eukaryotic hosts.
Metagenomics and virus discovery
In addition to the dsDNA phages typically thought to
dominate environmental viral communities, viral meta-
genomics has uncovered the presence of viral types not
previously described in certain environments (Table 2).
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 Environmental viral metagenomics Rosario and Breitbart 293

Evolutionary biologists can use genetic information from
novel viruses to investigate evolutionary links within viral
families as well as between viruses and their hosts
[35,41,44]. Single-stranded DNA (ssDNA) viruses are
gaining interest as metagenomic studies incorporating
multiple displacement amplification have recently
expanded the known host range and environmental
distribution of these viruses. Among ssDNA phages,
Microviridae have been identified in a wide range of
environments while Inoviridae have only been identified
in low abundance in a few viromes. Eukaryotic circular
ssDNA viruses were only known to infect plants, birds,
and mammals but their wide distribution in environmen-
tal viromes suggests that these viruses may infect a
broader range of hosts. This is supported by the recent
identification of circoviruses in invertebrates through

Table 2

DNA viral families identified in environmental samples and organisms through viral metagenomic approachesa

Viral family Environment or organism Known host Refs

Single-stranded DNA
Anelloviridae Harbor seals, Humans, Mosquitoes, Sea lions Mammals [66,67,48,68]
Gyrovirus genus Sea turtles Birds [69]
Circoviridae Antarctic lake, Man-made lake, Reclaimed water, Mammals/Birds [63,8,9,55,16,59,70,47,45,48]
British Columbia coastal waters, Chesapeake Bay,
Sargasso Sea, Rice paddy soil, Bats, Chimpanzees,
Dragonflies, Mosquitoes
Microviridae Antarctic lake, Reclaimed water, British Columbia Bacteria [63,9,7,6,34,59,70,26]
and California coastal waters, Gulf of Mexico,
Sargasso Sea, Tampa Bay, Rice paddy soil, Bats,
Freshwater and marine microbialites
Inoviridae Antarctic lake, Reclaimed and potable water, Bacteria [63,9,7,54,64]
Arctic Ocean, Marine sediment, Fermented food
Nanoviridae Antarctic lake, Reclaimed water, Strait of Georgia, Plants [63,9,55,34,59,47,60,48]
Tampa Bay, Rice paddy soil, Chimpanzees, Corals,
Mosquitoes
Geminiviridae Antarctic lake, Reclaimed water, Rice paddy soil, Plants [63,9,59,47,60,48]
Chimpanzees, Corals, Mosquitoes
Parvoviridae Bats, Corals, Mosquitoes, Turkeys, Fermented food Insects/Mammals [70,60,48,71,64]
Double-stranded DNA
Siphoviridae All DNA metagenomes Bacteria/Archaea –
Podoviridae All DNA metagenomes Bacteria –
Myoviridae All DNA metagenomes Bacteria/Archaea –
Rudiviridae Antarctic lake, Hot springs, Fermented food Archaea [63,23,64]
Fuselloviridae Hot springs, Fermented food Archaea [23,64]
Herpesviridae Antarctic lake, Reclaimed water, Hot springs, Mammals [63,9,23,7,60,61,48,64]
Arctic Ocean, Gulf of Mexico, Corals, Mosquitoes,
Fermented food
Mimiviridae Antarctic lake, Reclaimed water, Arctic Ocean, Algae/Protists [63,9,7,28,60,64]
Hydrothermal vent, Gulf of Mexico, Corals,
Fermented food
Phycodnaviridae Antarctic lake, Reclaimed water, Arctic Ocean, Algae [63,9,7,56,28,60,61,64]
Chesapeake Bay, Hydrothermal vent, Corals,
Fermented food
Iridoviridae Antarctic lake, Reclaimed water, British Columbia Fish/Amphibians/Insects [63,9,7,28,60,48,64]
coastal waters, Hydrothermal vent, Corals,
Mosquitoes, Fermented food
Poxviridae Antarctic lake, Reclaimed water, Hydrothermal vent, Mammals/Insects [63,9,7,28,70,60,72,48]
Bats, Corals, Horses, Mosquitoes
Nimaviridae Antarctic lake, Arctic Ocean, Corals Crustaceans [63,7,60]
Adenoviridae Bats, Corals, Fermented food Mammals [70,60,7]
Asfarviridae Antarctic lake, Arctic Ocean, Corals Mammals [63,7,60]
Polydnaviridae Antarctic lake, Corals Insects [63,60]
Baculoviridae Antarctic lake, Arctic Ocean, Corals Insects/Crustaceans [63,7,60]
Papillomaviridae Arctic Ocean, Corals, Mosquitoes Mammals [7,60,48]
Lipothrixviridae Antarctic lake Archaea [63]
Plasmaviridae Antarctic lake Bacteria [63]
Ascoviridae Antarctic lake, Fermented food Insects [63,64]
Tectiviridae Rice paddy soil, Fermented food Bacteria [59,64]
Caulimoviridae Corals, Fermented food Plants [60,64]
a
Table does not include viral families identified in tissues from humans exhibiting disease symptoms.

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294 Viral genomics

metagenomics ([45], Dunlap et al., unpublished data).
The discovery of genomes exhibiting mixed character-
istics from different ssDNA viral families [16,47,48] in
metagenomic data suggests that the evolutionary history
among eukaryotic ssDNA viruses may be more compli-
cated than previously recognized [46].
Metagenomics has also resulted in the discovery of RNA
viruses in the environment (Table 3). The few published
RNA viral metagenomes (from coastal waters, a man-
made lake, reclaimed water, humans, bats, and turkey
guts) suggest that positive-sense eukaryotic ssRNA
viruses dominate the RNA viral community regardless
of the environment. This mirrors the nonuniform distri-
bution of known RNA viruses where positive-sense
ssRNA viruses are more diverse (encompassing 108
genera and 27 families) than the other types of RNA
viruses [49]. The diversity of positive-sense RNA
viruses, in particular the picorna-like viruses, has been
explained by a potential early diversification of these
viruses predating the divergence of eukaryotic super-
groups [44].
Concluding remarks
Viral metagenomics has been instrumental for under-
standing viral ecology and exciting advances in this field
will benefit numerous disciplines (Figure 1). Scientists
are now developing functional viral metagenomics to
discover novel viral enzymes that can be used for diag-
nostic and biotechnological purposes [50]. Although this
field focuses on the discovery of enzymes with clinical or
commercial importance, it will likely improve the annota-
tion of viral genes with unknown functions since specific
functions are identified directly from the virome without
relying on similarities in existing databases [51]. Sur-
veillance for viral pathogens of importance for public
health and food security is another area that can take
advantage of viral metagenomics. Targeting viruses in
insect vectors known to transmit human and plant dis-
eases through vector-enabled metagenomics allows for

Table 3

RNA viral families identified in environmental samples and organisms through viral metagenomic approachesa

Viral family Environment or organism Known host Refs

Positive (+) and negative ( ) sense single-stranded RNA
+ Retroviridae Arctic Ocean, Corals, Fermented food Birds/Mammals [7,60,64]
Comovirinae Man-made lake, Reclaimed water, English Bay Plants [8,9,55]
Dicistroviridae Man-made lake, Reclaimed water, English Bay, Insects [8,9,55,70]
Strait of Georgia, Bats
Marnaviridae Man-made lake, English Bay Protists [8,55]
Picornaviridae Man-made lake, Reclaimed water, English Bay, Birds/Mammals [8,9,55,70,73,71]
Bats, Ringed seal, Turkeys
Sequiviridae Man-made lake, Reclaimed water Plants [8,9]
Iflavirus Man-made lake, Bats Insects [8,70]
Tombusviridae Man-made lake, Reclaimed water, Plants [8,9,55,74]
Strait of Georgia, Humans
Umbravirus Man-made lake, Strait of Georgia Plants [8,55]
Nodaviridae Man-made lake, Bats Fish/Insects [8,70]
Sobemovirus Man-made lake, Bats, Humans Plants [8,70,74]
Hepeviridae Man-made lake Birds/Mammals [8]
Flaviviridae Man-made lake Mammals [8]
Virgaviridae Man-made lake, Reclaimed water, Humans Plants [8,9,74]
Flexiviridae Man-made lake Plants [8]
Potyviridae Reclaimed water Plants [9]
Nora virus Man-made lake, Reclaimed water Insects [8,9]
Leviviridae Man-made lake, Turkeys Bacteria [8,71]
Tymoviridae Man-made lake, Bats, Humans, Turkeys Plants [8,70,74,71]
Tetraviridae Man-made lake, Bats Insects [8,70]
Astroviridae Bats, Turkeys Birds/Mammals [70,71]
Luteoviridae Man-made lake, Bats Plants [8,70]
Secoviridae Bats Plants [8]
Coronaviridae Arctic Ocean, Bats Birds/Mammals [7,70]
Bromoviridae Man-made lake, Humans Plants [8,74]
Orthomyxoviridae Man-made lake Birds/Mammals [8]
Double-stranded RNA
Picobirnavirus Man-made lake, Humans, Turkeys Mammals [8,74,71]
Reoviridae Man-made lake, Reclaimed water, English Bay Birds/Mammals/Insects/ [8,9,55]
Plants/Fungi
Partiviridae Man-made lake, Bats Plants/Protists/Fungi [8,70]
a
Table does not include viral families identified in tissues from humans exhibiting disease symptoms.

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surveillance of the viruses circulating in a given area and
provides a preemptive strategy for identifying emerging
pathogens [48,52]. Furthermore, metagenomic data
from human feces have been used to identify novel
bioindicators of fecal pollution with applications for
monitoring water quality [9,53]. Overall, viral metage-
nomics has provided the scientific community with tools
to tap into the massive environmental virome which holds
the key for elucidating viral diversity, understanding viral
evolution, and developing assays with important societal
applications.
Acknowledgements
This research was supported by grants to MB from the National Science
Foundation (DEB-1025915, OCE-1049670) and the Department of Energy
Genomes to Life Program. The authors would like to thank Yahayra
Rosario-Cora for the illustration in Figure 1.
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15. Tucker KP, Parsons R, Symonds EM, Breitbart M: Diversity and
distribution of single-stranded DNA phages in the North
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16. Rosario K, Duffy S, Breitbart M: Diverse circovirus-like genome
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17. Culley AI, Lang AS, Suttle CA: The complete genomes of three
viruses assembled from shotgun libraries of marine RNA virus
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18. Skennerton CT, Angly FE, Breitbart M, Bragg L, He S,
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potential achilles heel in the bacterial defence system. PLoS
One 2011, 6:e20095.
Assembly of viral metagenomic data led to the discovery of a phage
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19. Pride DT, Schoenfeld T: Genome signature analysis of thermal
virus metagenomes reveals Archaea and thermophilic
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20. Kapoor A, Simmonds P, Lipkin WI, Zaidi S, Delwart E: Use of
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21. Santos F, Yarza P, Parro V, Briones C, Anton J: The metavirome
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22. Breitbart M, Rohwer F: Here a virus, there a virus, everywhere
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23. Schoenfeld T, Patterson M, Richardson PM, Wommack KE,
Young M, Mead D: Assembly of viral metagenomes from
Yellowstone hot springs. Appl Environ Microbiol 2008,
74:4164-4174.
24. Sime-Ngando T, Lucas S, Robin A, Tucker KP, Colombet J,
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25. Fierer N, Breitbart M, Nulton J, Salamon P, Lozupone C, Jones R,
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Metagenomic and small-subunit rRNA analyses reveal the
genetic diversity of bacteria, archaea, fungi, and viruses in
soil. Appl Environ Microbiol 2007, 73:7059-7066.
26. Desnues C, Rodriguez-Brito B, Rayhawk S, Kelley S, Tran T,
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and biogeography of phages in modern stromatolites and
thrombolites. Nature 2008, 452:340-343.
27. Horvath P, Barrangou R: CRISPR/Cas, the immune system of
bacteria and archaea. Science 2010, 327:167-170.
28. Anderson RE, Brazelton WJ, Baross JA: Using CRISPRs as a
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hydrothermal vent viral assemblage. FEMS Microbiol Ecol 2011
doi: 10.1111/j.1574-6941.2011.01090.x.
Used a database of CRISPR spacers from all available prokaryotic
genomes to identify potential hosts for viruses sequenced from hydro-
thermal vents.
29. Andersson AF, Banfield JF: Virus population dynamics and
acquired virus resistance in natural microbial communities.
Science 2008, 320:1047-1050.
30. Kunin V, He S, Warnecke F, Peterson SB, Martin HG, Haynes M,
Ivanova N, Blackall LL, Breitbart M, Rohwer F et al.: A bacterial
metapopulation adapts locally to phage predation despite
global dispersal. Genome Res 2008, 18:293-297.
31. Heidelberg JF, Nelson WC, Schoenfeld T, Bhaya D: Germ warfare
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296 Viral genomics
32. Paul JH: Prophages in marine bacteria: dangerous molecular
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33. Williamson SJ, Rusch DB, Yooseph S, Halpern AL, Heidelberg KB,
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et al.: The Sorcerer II Global Ocean Sampling Expedition:
metagenomic characterization of viruses within aquatic
microbial samples. PLoS One 2008, 3:e1456.
34. McDaniel L, Breitbart M, Mobberley J, Long A, Haynes M,
Rohwer F, Paul JH: Metagenomic analysis of lysogeny in
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One 2008, 3:e3263.
35. Kristensen DM, Mushegian AR, Dolja VV, Koonin EV: New
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metagenomics. Trends Microbiol 2010, 18:11-19.
36. Lang AS, Beatty JT: Importance of widespread gene transfer
agent genes in alpha-proteobacteria. Trends Microbiol 2007,
15:54-62.
37. Breitbart M, Thompson LR, Suttle CA, Sullivan MB: Exploring the
vast diversity of marine viruses. Oceanography 2007,
20:135-139.
38. Monier A, Pagarete A, de Vargas C, Allen MJ, Read B, Claverie JM,
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pathway between a eukaryotic alga and its DNA virus. Genome
Res 2009, 19:1441-1449.
Demonstrated the horizontal gene transfer of seven genes that may be
involved in sphingolipid biosynthesis, providing the first clear example of
the transfer of an entire pathway between phytoplankton and viruses.
39. Sharon I, Battchikova N, Aro E-M, Giglione C, Meinnel T, Glaser F,
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metagenomics of microbial traits within oceanic viral
communities. ISME J 2011, 5:1178-1190.
Developed a strategy to identify viral scaffolds containing microbial genes
and detected a diversity of auxiliary metabolic genes amongst marine
metagenomes.
40. Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M, Brulc JM,
Furlan M, Desnues C, Haynes M, Li L et al.: Functional
metagenomic profiling of nine biomes. Nature 2008,
452:629-632.
41. Monier A, Claverie JM, Ogata H: Taxonomic distribution of large
DNA viruses in the sea. Genome Biol 2008, 9:R106.
42. Liu H, Fu Y, Jiang D, Li G, Xie J, Cheng J, Peng Y, Ghabrial SA, Yi X:
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43. Moreira D: Multiple independent horizontal transfers of
informational genes from bacteria to plasmids and phages:
implications for the origin of bacterial replication machinery.
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44. Koonin EV, Wolf YI, Nagasaki K, Dolja VV: The Big Bang of
picorna-like virus evolution antedates the radiation
of eukaryotic supergroups. Nat Rev Microbiol 2008,
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45. Rosario K, Marinov M, Stainton D, Kraberger S, Wiltshire EJ,
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Dragonfly cyclovirus, a novel single-stranded DNA virus
discovered in dragonflies (Odonata: Anisoptera). J Gen Virol
2011, 92:1302-1308.
First report of a single-stranded DNA circovirus in invertebrates, identified
using a viral metagenomic approach.
46. Gibbs MJ, Weiller GF: Evidence that a plant virus switched
hosts to infect a vertebrate and then recombined with a
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47. Blinkova O, Victoria J, Li Y, Keele BF, Sanz C, Ndjango J-BN,
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48. Ng TFF, Willner DL, Lim YW, Schmieder R, Chau B, Nilsson C,
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Current Opinion in Virology 2011, 1:289–297
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mosquitoes. PLoS One 2011, 6:e20579.
49. Hulo C, de Castro E, Masson P, Bougueleret L, Bairoch A, Xenarios I,
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50. Schoenfeld T, Liles M, Wommack KE, Polson SW, Godiska R,
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51. Schmitz JE, Schuch R, Fischetti VA: Identifying active phage
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Represents one of the first studies screening a viral metagenome for a
specific function resulting in the discovery of novel lysins.
52. Ng TFF, Duffy S, Polston JE, Bixby E, Vallad GE, Breitbart M:
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using vector-enabled metagenomics on whiteflies. PLoS One
2011, 6:e19050.
Proposed vector-enabled metagenomics as an approach to survey
vector-borne viruses circulating in a given region, and provided proof-
of-concept by examining plant viruses from whiteflies.
53. Rosario K, Symonds E, Sinigalliano C, Stewart J, Breitbart M:
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Environ Microbiol 2009, 75:7261-7267.
Building upon a viral metagenomic study that identified a plant virus
dominating the RNA viral community in human feces, this study deter-
mined that the plant virus can serve as a novel bioindicator of water
quality.
54. Breitbart M, Felts B, Kelley S, Mahaffy JM, Nulton J, Salamon P,
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marine-sediment viral community. Proc R Soc Lond Ser B-Biol
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55. Culley AI, Lang AS, Suttle CA: Metagenomic analysis of coastal
RNA virus communities. Science 2006, 312:1795-1798.
56. Bench SR, Hanson TE, Williamson KE, Ghosh D, Radosovich M,
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Chesapeake Bay virioplankton. Appl Environ Microbiol 2007,
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57. Rodriguez-Brito B, Li L, Wegley L, Furlan M, Angly F, Breitbart M,
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microbial community dynamics in four aquatic environments.
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58. Santos F, Meyerdierks A, Pena A, Rossello-Mora R, Amann R,
Anton J: Metagenomic approach to the study of halophages:
the environmental halophage 1. Environ Microbiol 2007,
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59. Kim KH, Chang HW, Nam YD, Roh SW, Kim MS, Sung Y, Jeon CO,
Oh HM, Bae JW: Amplification of uncultured single-stranded
DNA viruses from rice paddy soil. Appl Environ Microbiol 2008,
74:5975-5985.
60. Vega Thurber RL, Barott KL, Hall D, Liu H, Rodriguez-Mueller B,
Desnues C, Edwards RA, Haynes M, Angly FE, Wegley L et al.:
Metagenomic analysis indicates that stressors induce
production of herpes-like viruses in the coral Porites
compressa. Proc Nat Acad Sci U S A 2008, 105:18413-18418.
61. Marhaver KL, Edwards RA, Rohwer F: Viral communities
associated with healthy and bleaching corals. Environ
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62. Williamson SJ, Cary SC, Williamson KE, Helton RR, Bench SR,
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predominate at deep-sea diffuse-flow hydrothermal vents.
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63. Lopez-Bueno A, Tamames J, Velazquez D, Moya A, Quesada A,
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Antarctic lake. Science 2009, 326:858-861.
64. Park EJ, Kim KH, Abell GC, Kim MS, Roh SW, Bae JW:
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foods. Appl Environ Microbiol 2011, 77:1284-1291.
65. Parsley LC, Consuegra EJ, Thomas SJ, Bhavsar J, Land AM,
Bhuiyan NN, Mazher MA, Waters RJ, Wommack KE, Harper WF
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76:2673-2677.
66. Ng TF, Wheeler E, Greig D, Waltzek TB, Gulland F, Breitbart M:
Metagenomic identification of a novel anellovirus in Pacific
harbor seal (Phoca vitulina richardsii) lung samples and its
detection in samples from multiple years. J Gen Virol 2011,
92:1318-1323.
67. Breitbart M, Rohwer F: Method for discovering novel DNA
viruses in blood using viral particle selection and shotgun
sequencing. Biotechniques 2005, 39:729-736.
68. Ng TFF, Suedmeyer WK, Wheeler E, Gulland F, Breitbart M: Novel
anellovirus discovered from a mortality event of captive
California sea lions. J Gen Virol 2009, 90:1256-1261.
69. Ng TFF, Manire C, Borrowman K, Langer T, Ehrhart L, Breitbart M:
Discovery of a novel single-stranded DNA virus from a sea
turtle fibropapilloma by using viral metagenomics. J Virol 2009,
83:2500-2509.
www.sciencedirect.com
Environmental viral metagenomics Rosario and Breitbart 297
70. Li L, Victoria JG, Wang C, Jones M, Fellers GM, Kunz TH,
Delwart E: Bat guano virome: predominance of dietary viruses
from insects and plants plus novel mammalian viruses. J Virol
2010, 84:6955-6965.
71. Day JM, Ballard LL, Duke MV, Scheffler BE, Zsak L: Metagenomic
analysis of the turkey gut RNA virus community. Virol J 2010,
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72. Cann AJ, Fandrich SE, Heaphy S: Analysis of the virus
population present in equine faeces indicates the presence of
hundreds of uncharacterized virus genomes. Virus Genes 2005,
30:151-156.
73. Kapoor A, Victoria J, Simmonds P, Wang C, Shafer RW, Nims R,
Nielsen O, Delwart E: A highly divergent picornavirus in a
marine mammal. J Virol 2008, 82:311-320.
74. Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SWL,
Hibberd ML, Liu ET, Rohwer F, Ruan YJ: RNA viral community in
human feces: prevalence of plant pathogenic viruses. PLoS
Biol 2006, 4:108-118.
Current Opinion in Virology 2011, 1:289–297