Viral diversity and lifecycles are poorly understood in the human gut and other body habitats. Therefore, we sequenced the viromes (metagenomes) of virus-like particles isolated from fecal samples collected from adult female monozygotic twins and their mothers at three time points over a one-year period. These datasets were compared to datasets of sequenced bacterial 16S rRNA genes and total fecal community DNA. Co-twins and their mothers share a significantly greater degree of similarity in their fecal bacterial communities than do unrelated individuals. In contrast, viromes are unique to individuals regardless of their degree of genetic relatedness. Despite remarkable interpersonal variations in viromes and their encoded functions, intrapersonal diversity is very low, with >95% of virotypes retained over the period surveyed, and with viromes dominated by a few temperate phage that exhibit remarkable genetic stability. These results indicate that a predatory viral-microbial dynamic, manifest in a number of other characterized environmental ecosystems, is notably absent in the very distal intestine.
Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage-oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogenetically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut-specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative ‘viral-enterotypes’ among this fraction of the human gut virome.
Bacteriophages have a significant impact on microbial ecosystems, but additional tools are needed to assess viral communities. Ogilvie et al. present a new strategy to extract viral sequences from metagenomic data sets, and present new insights on their function in the gut ecosystem.
The aim of this study was to develop and demonstrate an approach for describing the diversity of human pathogenic viruses in an environmentally isolated viral metagenome.
Methods and Results
In silico bioinformatic experiments were used to select an optimum annotation strategy for discovering human viruses in virome datasets, and applied to annotate a class B biosolids virome. Results from the in silico study indicated that less than 1% errors in virus identification could be achieved when nucleotide-based search programs (BLASTn or tBLASTx), viral genome only databases, and sequence reads greater than 200 nt were considered. Within the 51,925 annotated sequences, 94 DNA and 19 RNA sequences were identified as human viruses. Virus diversity included environmentally transmitted agents such as parechovirus, coronavirus, adenovirus, and aichi virus, as well as viruses associated with chronic human infections such as human herpes and hepatitis C viruses.
This study provided a bioinformatic approach for identifying pathogens in a virome dataset, and demonstrated the human virus diversity in a relevant environmental sample.
Significance and Impact of Study
As the costs of next generation sequencing decrease, the pathogen diversity described by virus metagenomes will provide an unbiased guide for subsequent cell-culture and quantitative pathogen analyses, and ensures that highly enriched and relevant pathogens are not neglected in exposure and risk assessments.
virus; bioinformatics; biosolids; next generation DNA sequencing; viral metagenome; pathogen; virome
California sea lions are one of the major marine mammal species along the Pacific coast of North America. Sea lions are susceptible to a wide variety of viruses, some of which can be transmitted to or from terrestrial mammals. Using an unbiased viral metagenomic approach, we surveyed the fecal virome in California sea lions of different ages and health statuses. Averages of 1.6 and 2.5 distinct mammalian viral species were shed by pups and juvenile sea lions, respectively. Previously undescribed mammalian viruses from four RNA virus families (Astroviridae, Picornaviridae, Caliciviridae, and Reoviridae) and one DNA virus family (Parvoviridae) were characterized. The first complete or partial genomes of sapeloviruses, sapoviruses, noroviruses, and bocavirus in marine mammals are reported. Astroviruses and bocaviruses showed the highest prevalence and abundance in California sea lion feces. The diversity of bacteriophages was higher in unweaned sea lion pups than in juveniles and animals in rehabilitation, where the phage community consisted largely of phages related to the family Microviridae. This study increases our understanding of the viral diversity in marine mammals, highlights the high rate of enteric viral infections in these highly social carnivores, and may be used as a baseline viral survey for comparison with samples from California sea lions during unexplained disease outbreaks.
Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics.
next generation sequencing; deep sequencing; virus discovery; metagenomics; virome; virology; quasispecies; molecular diagnosis; human immunodeficiency virus; drug resistance; minority variants
Phylogenetic mapping of metagenomics data reveals the taxonomic distribution of large DNA viruses in the sea, including giant viruses of the Mimiviridae family.
Viruses are ubiquitous and the most abundant biological entities in marine environments. Metagenomics studies are increasingly revealing the huge genetic diversity of marine viruses. In this study, we used a new approach - 'phylogenetic mapping' - to obtain a comprehensive picture of the taxonomic distribution of large DNA viruses represented in the Sorcerer II Global Ocean Sampling Expedition metagenomic data set.
Using DNA polymerase genes as a taxonomic marker, we identified 811 homologous sequences of likely viral origin. As expected, most of these sequences corresponded to phages. Interestingly, the second largest viral group corresponded to that containing mimivirus and three related algal viruses. We also identified several DNA polymerase homologs closely related to Asfarviridae, a viral family poorly represented among isolated viruses and, until now, limited to terrestrial animal hosts. Finally, our approach allowed the identification of a new combination of genes in 'viral-like' sequences.
Albeit only recently discovered, giant viruses of the Mimiviridae family appear to constitute a diverse, quantitatively important and ubiquitous component of the population of large eukaryotic DNA viruses in the sea.
Nearly complete genome sequences of three novel RNA viruses were acquired from the stool of an Afghan child. Phylogenetic analysis indicated that these viruses belong to the picorna-like virus superfamily. Because of their unique genomic organization and deep phylogenetic roots, we propose these viruses, provisionally named calhevirus, tetnovirus-1, and tetnovirus-2, as prototypes of new viral families. A newly developed nucleotide composition analysis (NCA) method was used to compare mononucleotide and dinucleotide frequencies for RNA viruses infecting mammals, plants, or insects. Using a large training data set of 284 representative picornavirus-like genomic sequences with defined host origins, NCA correctly identified the kingdom or phylum of the viral host for >95% of picorna-like viruses. NCA predicted an insect host origin for the 3 novel picorna-like viruses. Their presence in human stool therefore likely reflects ingestion of insect-contaminated food. As metagenomic analyses of different environments and organisms continue to yield highly divergent viral genomes NCA provides a rapid and robust method to identify their likely cellular hosts.
Acanthamoeba polyphaga mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the Acanthamoeba genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data.
We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (Chrysochromulina ericina, Phaeocystis pouchetii, Pyramimonas orientalis) are very closely related to their homolog in mimivirus.
Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of Mimiviridae.
Viruses are the most abundant known infectious agents on the planet and are significant drivers of diversity in a variety of ecosystems. Although there have been numerous studies of viral communities, few have focused on viruses within the indigenous human microbiota. We analyzed 2 267 695 virome reads from viral particles and compared them with 263 516 bacterial 16S rRNA gene sequences from the saliva of five healthy human subjects over a 2- to 3-month period, in order to improve our understanding of the role viruses have in the complex oral ecosystem. Our data reveal viral communities in human saliva dominated by bacteriophages whose constituents are temporally distinct. The preponderance of shared homologs between the salivary viral communities in two unrelated subjects in the same household suggests that environmental factors are determinants of community membership. When comparing salivary viromes to those from human stool and the respiratory tract, each group was distinct, further indicating that habitat is of substantial importance in shaping human viromes. Compared with coexisting bacteria, there was concordance among certain predicted host–virus pairings such as Veillonella and Streptococcus, whereas there was discordance among others such as Actinomyces. We identified 122 728 virulence factor homologs, suggesting that salivary viruses may serve as reservoirs for pathogenic gene function in the oral environment. That the vast majority of human oral viruses are bacteriophages whose putative gene function signifies some have a prominent role in lysogeny, suggests these viruses may have an important role in helping shape the microbial diversity in the human oral cavity.
saliva; bacteriophage; virus; microbiome; virome; metagenome
In this study, we analyzed viral metagenomes (viromes) in the sedimentary habitats of three geographically and geologically distinct (hado)pelagic environments in the northwest Pacific; the Izu-Ogasawara Trench (water depth = 9,760 m) (OG), the Challenger Deep in the Mariana Trench (10,325 m) (MA), and the forearc basin off the Shimokita Peninsula (1,181 m) (SH). Virus abundance ranged from 106 to 1011 viruses/cm3 of sediments (down to 30 cm below the seafloor [cmbsf]). We recovered viral DNA assemblages (viromes) from the (hado)pelagic sediment samples and obtained a total of 37,458, 39,882, and 70,882 sequence reads by 454 GS FLX Titanium pyrosequencing from the virome libraries of the OG, MA, and SH (hado)pelagic sediments, respectively. Only 24−30% of the sequence reads from each virome library exhibited significant similarities to the sequences deposited in the public nr protein database (E-value <10−3 in BLAST). Among the sequences identified as potential viral genes based on the BLAST search, 95−99% of the sequence reads in each library were related to genes from single-stranded DNA (ssDNA) viral families, including Microviridae, Circoviridae, and Geminiviridae. A relatively high abundance of sequences related to the genetic markers (major capsid protein [VP1] and replication protein [Rep]) of two ssDNA viral groups were also detected in these libraries, thereby revealing a high genotypic diversity of their viruses (833 genotypes for VP1 and 2,551 genotypes for Rep). A majority of the viral genes predicted from each library were classified into three ssDNA viral protein categories: Rep, VP1, and minor capsid protein. The deep-sea sedimentary viromes were distinct from the viromes obtained from the oceanic and fresh waters and marine eukaryotes, and thus, deep-sea sediments harbor novel viromes, including previously unidentified ssDNA viruses.
There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.
Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.
Swine are an important source of proteins worldwide but are subject to frequent viral outbreaks and numerous infections capable of infecting humans. Modern farming conditions may also increase viral transmission and potential zoonotic spread. We describe here the metagenomics-derived virome in the feces of 24 healthy and 12 diarrheic piglets on a high-density farm. An average of 4.2 different mammalian viruses were shed by healthy piglets, reflecting a high level of asymptomatic infections. Diarrheic pigs shed an average of 5.4 different mammalian viruses. Ninety-nine percent of the viral sequences were related to the RNA virus families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae, while 1% were related to the small DNA virus families Circoviridae, and Parvoviridae. Porcine RNA viruses identified, in order of decreasing number of sequence reads, consisted of kobuviruses, astroviruses, enteroviruses, sapoviruses, sapeloviruses, coronaviruses, bocaviruses, and teschoviruses. The near-full genomes of multiple novel species of porcine astroviruses and bocaviruses were generated and phylogenetically analyzed. Multiple small circular DNA genomes encoding replicase proteins plus two highly divergent members of the Picornavirales order were also characterized. The possible origin of these viral genomes from pig-infecting protozoans and nematodes, based on closest sequence similarities, is discussed. In summary, an unbiased survey of viruses in the feces of intensely farmed animals revealed frequent coinfections with a highly diverse set of viruses providing favorable conditions for viral recombination. Viral surveys of animals can readily document the circulation of known and new viruses, facilitating the detection of emerging viruses and prospective evaluation of their pathogenic and zoonotic potentials.
Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.
RNA viruses; DNA viruses; virioplankton; marine; seawater; abundance; metagenome
The human virome is the collection of all viruses that are found in or on humans, including both eukaryotic and prokaryotic viruses. Eukaryotic viruses clearly have important effects on human health, ranging from mild, self-limited acute or chronic infections to those with serious or fatal consequences. Prokaryotic viruses can also affect human health by impacting bacterial community structure and function. Therefore, definition of the virome is an important step toward understanding how microbes affect human health and disease. We review progress in virome analysis, which has been driven by advances in high-throughput, deep sequencing technology. Highlights from these studies include the association of viruses with clinical phenotypes and description of novel viruses that may be important pathogens. Together these studies indicate that analysis of the human virome is critical as we aim to understand how microbial communities affect human health and disease. Descriptions of the human virome will stimulate future work to understand how the virome affects long-term human health, immunity, and response to co-infections. Ultimately analysis of the virome may affect the treatment of patients with a variety of clinical syndromes.
Recent advances of genomics and metagenomics reveal remarkable diversity of viruses and other selfish genetic elements. In particular, giant viruses have been shown to possess their own mobilomes that include virophages, small viruses that parasitize on giant viruses of the Mimiviridae family, and transpovirons, distinct linear plasmids. One of the virophages known as the Mavirus, a parasite of the giant Cafeteria roenbergensis virus, shares several genes with large eukaryotic self-replicating transposon of the Polinton (Maverick) family, and it has been proposed that the polintons evolved from a Mavirus-like ancestor.
We performed a comprehensive phylogenomic analysis of the available genomes of virophages and traced the evolutionary connections between the virophages and other selfish genetic elements. The comparison of the gene composition and genome organization of the virophages reveals 6 conserved, core genes that are organized in partially conserved arrays. Phylogenetic analysis of those core virophage genes, for which a sufficient diversity of homologs outside the virophages was detected, including the maturation protease and the packaging ATPase, supports the monophyly of the virophages. The results of this analysis appear incompatible with the origin of polintons from a Mavirus-like agent but rather suggest that Mavirus evolved through recombination between a polinton and an unknownvirus. Altogether, virophages, polintons, a distinct Tetrahymena transposable element Tlr1, transpovirons, adenoviruses, and some bacteriophages form a network of evolutionary relationships that is held together by overlapping sets of shared genes and appears to represent a distinct module in the vast total network of viruses and mobile elements.
The results of the phylogenomic analysis of the virophages and related genetic elements are compatible with the concept of network-like evolution of the virus world and emphasize multiple evolutionary connections between bona fide viruses and other classes of capsid-less mobile elements.
RNA viruses have been isolated that infect marine organisms ranging from bacteria to whales, but little is known about the composition and population structure of the in situ marine RNA virus community. In a recent study, the majority of three genomes of previously unknown positive-sense single-stranded (ss) RNA viruses were assembled from reverse-transcribed whole-genome shotgun libraries. The present contribution comparatively analyzes these genomes with respect to representative viruses from established viral taxa.
Two of the genomes (JP-A and JP-B), appear to be polycistronic viruses in the proposed order Picornavirales that fall into a well-supported clade of marine picorna-like viruses, the characterized members of which all infect marine protists. A temporal and geographic survey indicates that the JP genomes are persistent and widespread in British Columbia waters. The third genome, SOG, encodes a putative RNA-dependent RNA polymerase (RdRp) that is related to the RdRp of viruses in the family Tombusviridae, but the remaining SOG sequence has no significant similarity to any sequences in the NCBI database.
The complete genomes of these viruses permitted analyses that resulted in a more comprehensive comparison of these pathogens with established taxa. For example, in concordance with phylogenies based on the RdRp, our results support a close homology between JP-A and JP-B and RsRNAV. In contrast, although classification of the SOG genome based on the RdRp places SOG within the Tombusviridae, SOG lacks a capsid and movement protein conserved within this family and SOG is thus likely more distantly related to the Tombusivridae than the RdRp phylogeney indicates.
Viral genomes often contain genes recently acquired from microbes. In some cases (for example, psbA) the proteins encoded by these genes have been shown to be important for viral replication. In this study, using a unique search strategy on the Global Ocean Survey (GOS) metagenomes in combination with marine virome and microbiome pyrosequencing-based datasets, we characterize previously undetected microbial metabolic capabilities concealed within the genomes of uncultured marine viral communities. A total of 34 microbial gene families were detected on 452 viral GOS scaffolds. The majority of auxiliary metabolic genes found on these scaffolds have never been reported in phages. Host genes detected in viruses were mainly divided between genes encoding for different energy metabolism pathways, such as electron transport and newly identified photosystem genes, or translation and post-translation mechanism related. Our findings suggest previously undetected ways, in which marine phages adapt to their hosts and improve their fitness, including translation and post-translation level control over the host rather than the already known transcription level control.
cyanophage; gene transfer; metagenomics; photosynthesis; viral–host interactions
Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes.
Bacteriophages or phages are the most abundant organisms in the biosphere and they are a ubiquitous feature of prokaryotic existence. A bacteriophage is a virus which infects a bacterium. Archaea are also infected by viruses, whether these should be referred to as ‘phages’ is debatable, but they are included as such in the scope this article. Phages have been of interest to scientists as tools to understand fundamental molecular biology, as vectors of horizontal gene transfer and drivers of bacterial evolution, as sources of diagnostic and genetic tools and as novel therapeutic agents. Unraveling the biology of phages and their relationship with their hosts is key to understanding microbial systems and their exploitation. In this article we describe the roles of phages in different host systems and show how modeling, microscopy, isolation, genomic and metagenomic based approaches have come together to provide unparalleled insights into these small but vital constituents of the microbial world.
bacteriophage; ecology; cyanophages; archaeal viruses; animal microbiome
The human respiratory tract is heavily exposed to microorganisms. Viral respiratory tract pathogens, like RSV, influenza and rhinoviruses cause major morbidity and mortality from respiratory tract disease. Furthermore, as viruses have limited means of transmission, viruses that cause pathogenicity in other tissues may be transmitted through the respiratory tract. It is therefore important to chart the human virome in this compartment. We have studied nasopharyngeal aspirate samples submitted to the Karolinska University Laboratory, Stockholm, Sweden from March 2004 to May 2005 for diagnosis of respiratory tract infections. We have used a metagenomic sequencing strategy to characterize viruses, as this provides the most unbiased view of the samples. Virus enrichment followed by 454 sequencing resulted in totally 703,790 reads and 110,931 of these were found to be of viral origin by using an automated classification pipeline. The snapshot of the respiratory tract virome of these 210 patients revealed 39 species and many more strains of viruses. Most of the viral sequences were classified into one of three major families; Paramyxoviridae, Picornaviridae or Orthomyxoviridae. The study also identified one novel type of Rhinovirus C, and identified a number of previously undescribed viral genetic fragments of unknown origin.
The human body is colonized with a diverse resident microflora that includes viruses. Recent studies of metagenomes have begun to characterize the composition of the human ‘virobiota’ and its associated genes (the ‘virome’), and have fostered the emerging field of host-virobiota interactions. In this Perspective, we explore how resident viruses interact with the immune system. We review recent findings that highlight the role of the immune system in shaping the composition of the virobiota and consider how resident viruses may impact host immunity. Finally, we discuss the implications of virobiota–immune system interactions for human health.
The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome.
metagenome; marine; microorganism; enzyme
Viruses are an integral component of the marine food web, contributing to the disease and mortality of essentially every type of marine life, yet the diversity of viruses in the sea, especially those with RNA genomes, remains very poorly characterized. Isolates of RNA-containing viruses that infect marine plankton are still rare, and the only cultivation-independent surveys of RNA viral diversity reported so far were conducted for temperate coastal waters of British Columbia. Here, we report on our improvements to a previously used protocol to investigate the diversity of marine picorna-like viruses and our results from applying this protocol in subtropical waters. The original protocol was simplified by using direct filtration, rather than tangential flow filtration, to harvest viruses from seawater, and new degenerate primers were designed to amplify a fragment of the RNA-dependent RNA polymerase gene by reverse transcription-PCR from RNA extracted from the filters. Whereas the original protocol was unsuccessful in a preliminary test, the new protocol resulted in amplification of picorna-like virus sequences in every sample of subtropical and temperate coastal seawater assayed. These polymerase sequences formed a diverse, but monophyletic cluster along with other sequences amplified previously from seawater and sequences from isolates infecting marine protists. Phylogenetic analysis suggested that our sequences represent at least five new genera and 24 new species of RNA viruses. These results contribute to our understanding of RNA virus diversity and suggest that picorna-like viruses are a source of mortality for a wide variety of marine protists.
The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated.