viruses are the most abundant and diverse biological entities in the biosphere , and their global numbers have been estimated at 1.2  10 and 2.6  10 in the ocean and soil , respectively .
thus , viruses are key elements that contribute to the life cycles of cellular organisms . however , the study of viral ecology in natural habitats has been limited due to the difficulties of viral culture .
the lack of a ubiquitous marker gene , such as the 16s rrna gene shared by all bacteria and archaea , also hampered our understanding of the genetic diversity of viruses , prior to the introduction of metagenomics into the field of viral ecology [ 1 , 4 ] .
recently , viral metagenomics has enabled researchers to explore the community structure and diversity of viruses in various natural ecosystems .
this methodology depends on a priori knowledge of the viral types that may be present [ 6 , 7 ] .
the first viral metagenome of uncultured marine viral communities was published in 2002 , and there have been many subsequent advances in the methodologies ( e.g. , methods for amplifying the initial viral genomes ) and tools used for bioinformatics analysis in viral metagenomics .
these technical developments have facilitated explorations of the abundance and diversity of viruses from a wide range of natural habitats in korea .
in korea , viral metagenomics was applied for the first time to unique samples , including fermented foods and atmospheric samples , as well as habitats where viruses are expected to have significant roles , such as rice paddy soil , seawater , and the human gut .
this review describes recent advances in viral metagenomics and provides summaries of studies that have been conducted to characterize korean viral metagenomes .
in addition , the advantages and disadvantages of the most widely used viral dna amplification methods are discussed , based on empirical knowledge .
viral metagenomics is the study of viral metagenomes ( known as viromes ) , which are obtained directly from environmental samples using viral particle purification and shotgun sequencing .
viral metagenomic studies have increased gradually since the expansion of metagenomic approaches to viral ecology ; i.e. , over 200 ( 61 reviews ) investigations of the viral communities in environmental samples have been published ( fig .
1 ) [ 8 - 13 ] . in 2002 , breitbart et al .   used shotgun library sequencing and reported that the majority of the sequences in marine viral metagenomes shared no similarity with any genes in public databases , which suggested that most environmental viruses remained uncharacterized .
subsequently , many studies have surveyed the viral diversity of unexplored habitats using viral metagenomics .
cultivation in a host is usually necessary to obtain a virus from the environment that is being investigated .
however , the application of metagenomics techniques based on genetic information can circumvent this obstacle .
based on the physical characteristics of virions , viral particles can be isolated from environmental samples , which are enriched using a combination of size filtration ( e.g. , < 0.22 m ) and density gradient centrifugation ( e.g. , 1.35 - 1.5 g / ml cesium chloride ) , so that the virome can be obtained from the purified viral particles .
there is a lack of evolutionarily conserved genes , such as the prokaryotic 16s ribosomal rna gene , in viral genomes .
therefore , the fragmented viral metagenomic sequences obtained by whole - genome shotgun dna sequencing are used to analyze the viral ecology instead .
viral genomes are small ( on average , they comprise a few to several dozen kilobases ) ; so , valuable genome coverage can be achieved easily by dna sequencing .
the advent of high - throughput sequencing techniques , such as 454 pyrosequencing and illumina sequencing , makes it easier to achieve suitable genome coverage at low cost .
indeed , over 90 of either nearly complete or complete novel viral genomes were assembled using these methods in recent studies [ 9 , 10 , 15 - 20 ] .
however , it is inevitable that an amplification step is necessary for small viral genomes prior to dna sequencing .
linker amplified shotgun library ( lasl ) and multiple displacement amplification ( mda ) are the main methods used to amplify viromes in viral metagenomics .
the application of adapter attachment is restricted to double - stranded dna sequences ; so , only double - stranded dna ( dsdna ) viruses can be detected using the lasl method .
thus , the dominance of dsdna tailed bacteriophages was reported initially in uncultured marine viral assemblages , as well as other environmental samples , such as kimchi , aquatic water , and feces .
the mda technique   amplifies dna isothermally using the phi29 polymerase and random hexaprimers and has been used before in microbiology .
the high amplification efficiency of this method means that it is suitable for amplification during viral metagenomics applications .
particularly , the phi29 polymerase of the mda technique selectively amplifies circular genomes ( estimated at 100 times ) , and it has facilitated the discovery of abundant single - stranded dna and rna viruses in environmental samples [ 23 , 24 ] ( described in detail below ) .
the analysis of viral metagenome data using bioinformatics is one of the most challenging aspects of viral metagenomics .
one million to 1 billion reads of viral metagenomic sequences are typically generated by high - throughput sequencing platforms ( with an average read length of 350 - 400 bp using 454 gs - flx titanium and 2  150 bp using illumina hiseq 2500 ) . after removing any low - quality , redundant , and chimeric sequences ,
the viral sequences are compared to sequences in public databases ( e.g. , the genbank non - redundant nucleotide database , mg - rast , and camera ) using blast   or usearch .
the identification of viral sequences based on significant amino acid similarity ( e - value of < 10 ) was first described by breitbart et al . , and it has since been extended to the exploration of environmental viromes , although the e - score applied to viral metagenomic studies appears to be regarded as " a loose standard " .
most of the environmental viromes detected by viral metagenomics are defined as orphan ( unassigned ) sequences .
the majority of viral sequences shares no amino acid similarity with previously observed genes ( average 40% to 50% , occasionally up to 90% , of sequences ) ; so , they are characterized as " unknown " .
comparisons of viral sequences with the data in public databases have demonstrated that little is known about environmental viruses .
thus , the majority of the unassigned sequences in viral metagenomes is often regarded as " junk sequences " due to a lack of suitable bioinformatics tools and viral databases for their characterization [ 1 , 28 ] . at present , the viral databases are biased toward animal and plant viruses , although viruses that infect prokaryotes ( bacteriophages ) are sparsely represented . most of the latter are restricted to phages that infect bacteria belonging to the phyla proteobacteria , firmicutes , and actinobacteria [ 29 , 30 ] . moreover ,
even " known " viral sequences share low amino acid similarities ( < 50% ) with viral protein sequences [ 9 , 11 , 13 , 31 ] ; so , the majority of environmental viruses representing novel viral species and their viral diversity is much greater than considered previously .
the observation of a high percentage of orfans ( open reading frames with no homologs in known genes in the databases ) in viral genomes   also supports the novelty of environmental viromes .
thus , researchers could discover novel viruses in orphan sequence pools that currently remain " untapped resources . "
these findings indicate our current lack of knowledge about viral genetic information and emphasize the need for physiological studies of viruses to understand viral ecology based on genomic data .
using viral metagenomic approaches , viral diversity and abundance have been investigated in various natural ecosystems in korea , including rice paddy soil , fermented foods , the human gut , seawater , and the near - surface atmosphere ( table 1 ) .
2 ) . sipho- and podo - like tailed viruses were found in fermented foods ( fig .
2a-2c ) , while non - tailed small viruses were detected in the near - surface atmosphere ( fig .
various types of tailed , non - tailed , circular , and long linear viruses were found in the human gut ( fig .
in general , the virions ranged in size from 30 to 60 nm . in agreement with the results of previous viral metagenomic studies [ 23 , 31 ] , over half of the sequences in the korean environmental viromes were described as orphan sequences , based on comparisons with viral proteins in public databases ( table 1 ) .
most of the sequences identified were assigned to the siphoviridae , podoviridae , and myoviridae families of dsdna viruses and the circoviridae , germiniviridae , nanoviridae , and microviridae families of ssdna viruses ( fig .
the first viral metagenomic study in korea surveyed uncultured viral assemblages in rice paddy soil in 2008 , where mda was used with phi29 dna poly merase and random hexaprimers to amplify viral dna and to construct clone libraries for metagenome sequencing .
the soil was found to contain a rich pool of unknown ssdna viruses and dsdna viruses .
this study also focused on the effect of mda amplification on different types of genomic dna and showed that mda preferentially amplified circular dna genomes .
this was also demonstrated using an environmental sample from surface seawater , where dsdna viruses alone were retrieved in the lasl library , whereas ssdna viruses were overwhelmingly represented in the mda library .
thus , the amplification methods used in viral metagenomics can affect the ratios of viral sequences greatly and lead to inaccurate estimates of viral diversity .
next , park et al .   investigated the abundance and diversity of uncultured viral assemblages in fermented shrimp , kimchi , and sauerkraut - fermented foods that have been consumed for a long time around the world .
in contrast to the soil virome , dsdna bacteriophages from the families myoviridae , podoviridae , and siphoviridae dominated the fermented foods , and they contained a low complexity of viral assemblages compared with other environmental habitats , such as seawater , human feces , marine sediment , and soil .
however , it is possible that the viral diversity of the viromes detected in fermented foods may have been constrained to dsdna viruses by the lasl method .
a large number of unknown microbes , such as bacteria , archaea , microbial eukarya , and viruses , constitute up to 10 bacteria per gram of feces in the human gastrointestinal tract , and it is expected that gut viruses will affect the relationships among viruses , bacteria , and gut epithelial cells .
kim et al .   investigated the abundance and diversity of dna viruses in fecal samples from five healthy koreans , particularly ssdna viruses . using epifluorescence microscopy with sybr gold staining ,
the viral abundance ranged from 10 to 10 per gram of feces , which was 10-fold less than the bacterial abundance in many other environments that harbored 10 - 100-fold more viruses ( e.g. , aquatic environments ) . moreover , the diversity of gut viral assemblages was lower than that of gut bacteria .
who found that viral - microbial interactions in the human intestine could not be described as a predator - prey relationship , and instead , it was referred to as " kill the winner , " which was driven by a lytic life cycle .
airborne viruses are now regarded as major environmental risk factors for complex disease pathogenesis [ 36 - 38 ] .
however , the atmosphere remains " one of the last frontiers of biological exploration on earth " .
using viral metagenomics with an advanced airborne particle sampling system , whon et al .  
conducted the first study of the diversity and community composition of airborne viruses in the near - surface atmosphere .
the viral abundance in the atmosphere exhibited seasonal changes ( increasing from autumn to winter before decreasing until spring ) in the range of 10 to 10 viruses per m , and the temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity .
plant - associated ssdna geminivirus - related viruses and animal - infecting circoviruses dominated the viral assemblages , with low numbers of nanoviruses and microphages in air viromes , which suggests that airborne viral assemblages are affected greatly by terrestrial plants and animal activities .
thus , the compositions of the viral assemblages detected in fermented foods and marine samples are biased toward dsdna viruses , such as sipho- , podo- , and myophages , when the lasl method is used , whereas the viral assemblages detected in rice paddy soil , human gut , marine , and near - surface atmosphere samples contain high proportions of ssdna viral sequences , due to the use of mda , as shown in fig .
, the compositions of the viral assemblages characterized in korean environments tend to depend on their specific microbial features .
the human gastrointestinal tract and fermented foods are exposed to massive numbers of gut bacteria and lactic acid bacteria , respectively [ 39 - 41 ] . by contrast
, the atmosphere contains far less cellular metabolism and reproductive activity than other environments , such as the soil , seawater , fermented foods , and the human gut . on this basis ,
the lowest abundance of eukaryotic viruses was observed in the viral assemblages in the human gut and fermented foods , whereas a high abundance of eukaryotic viruses was detected in the near - surface atmosphere .
high levels of prokaryote and eukaryote cells are present in rice paddy soil   and seawater , and so , comparable amounts of bacteriophages and eukaryotic viruses were detected in their viromes .
the development of viral metagenomics has facilitated the discovery of novel , previously undescribed viral species .
an artifact of the mda method is that it selectively amplifies the circular genomes of ssdna viruses , so that a large number of ssdna viral sequences have been identified in environmental viromes .
thus , there is great interest in the distribution and host range of ssdna viruses .
in particular , microphages from the family microviridae have been identified in a wide range of environments [ 13 , 45 , 46 ] .
in contrast to the ecology of marine dsdna phages , marine ssdna phages in the family microviridae have distinct spatial and temporal distributions .
ssdna microphages were abundant in the healthy human gut and their genotypes were much more diverse than those reported previously .
moreover , prophage - like elements in the genomes of gut microbes , such as bacteroides and prevotella spp . , were characterized as a novel subgroup in the family microviridae [ 13 , 47 ] , while viral sequences from the human gut were clustered with prophage - like elements from bacteroides and prevotella spp . .
eukaryotic ssdna viruses that infect plants and mammals have been identified in many environmental viromes .
circoviruses that are known to infect birds and pigs have also been identified in the viromes of invertebrates and a fish [ 48 , 49 ] , while geminiviruses that cause plant diseases have been detected in whiteflies , which act as insect vectors of plant viruses .
a recent study by whon et al .   investigated airborne dna viral assemblages in near - surface atmosphere samples and showed that a high number of viruses ( log 6 to 7 viruses per m ) were present in the air , which were dominated by geminivirus - related viruses .
, sclerotinia sclerotiorum hypovirulence - associated dna virus 1-which indicates that the airborne viral assemblages in the near - surface atmosphere may have strong interactions with plants .
these results highlight the extensive distribution of ssdna viruses in a wide range of environments , and their host ranges may be wider than previously recognized .
thus , the discovery of novel genomes of ssdna viral families in metagenomic studies could revolutionize our knowledge of the ecology and evolution of ssdna viruses .
in the last decade , viral ecologists have focused on community - level analyses of viruses to understand their abundance and genetic diversity in specific environments .
the ecological effects of viruses , particularly bacteriophages , are known to control host populations via the " kill the winner system , " while they drive mortality and evolutionary change in microorganisms via lateral gene transfer by infecting their host bacteria , although the basic issue of " who infects whom " is poorly understood [ 2 , 29 , 51 , 52 ] . in the ocean , for example , viruses regulate the microbial abundance , release dissolved organic matter , and affect global biogeochemical cycles by killing up to 40% of host bacteria per day [ 53 , 54 ] .
in contrast , symbiotic functions of viruses , such as host survival , competition , and protection from pathogenic infections , are beginning to be understood , and evidence for a beneficial interaction in phage - host interactions was found in the mammalian gut ecosystem [ 29 , 56 ] .
when host survival is threatened , a variety of environmental factors can trigger prophage induction , and the liberated prophages may become completely virulent .
overall , these studies suggest that prophage induction may responsible for triggering dysbiosis and changes in the microbial population by altering host phenotypes , thereby leading to a new environmental niche .
traditionally , host culture - dependent techniques , such as plaque assays , have been widely used for the identification of phage and host bacteria interactions .
however , plaque assays require isolated host bacteria ; so , they are low - throughput methods .
this method is also difficult to apply to environmental samples where lysogenic infections are prevalent , because the method relies on observations of visible plaque formations , which are often absent from lysogenic infections [ 3 , 58 , 59 ] .
recently , deng et al .   demonstrated a new technique , known as " viral tagging , " for identifying the interactions between cultivated host bacteria and their phages , which used the nucleic acid stain sybr gold to generate fluorescently labeled phages , so that the host cells fluoresced with viral tagging , thereby allowing the sorting of virus - tagged cells by flow cytometry [ 52 , 60 ] .
this emerging technique is undoubtedly helpful for not only exploring virus - host interactions in their natural habitats when the method is combined with other experimental tools , such as single viral genomics   and phagefish , but also identifying viral receptors in macro - organisms ( e.g. , the mammalian gut ) if the method is combined with a fluorescently labeled receptor protein during histological examinations .
the emergence of viral metagenomics has facilitated advances in virology and allowed us to understand novel aspects of viral ecology . at present ,
viral metagenomics is a powerful and sensitive technique for detecting viruses that can not be identified by traditional culture- and sequence - based approaches .
most importantly , viral metagenomics suggests that novel viruses interact constantly with the human population .
thus , viral metagenomics can facilitate the improved surveillance of viral pathogens in the fields of public health and food security .
this technique can be used to understand viral ecology by exploring the environmental viromes that are generated by viral metagenomics .
 the introduction of metagenomics into the field of virology has facilitated the exploration of viral communities in various natural habitats . 
 understanding the viral ecology of a variety of sample types throughout the biosphere is important per se , but it also has potential applications in clinical and diagnostic virology . 
 however , the procedures used by viral metagenomics may produce technical errors , such as amplification bias , while public viral databases are very limited , which may hamper the determination of the viral diversity in samples . 
 this review considers the current state of viral metagenomics , based on examples from korean viral metagenomic studies - i.e . , rice paddy soil , fermented foods , human gut , seawater , and the near - surface atmosphere . 
 viral metagenomics has become widespread due to various methodological developments , and much attention has been focused on studies that consider the intrinsic role of viruses that interact with their hosts . 