Metagenomics, or sequencing of the genetic material from a complete microbial community, is a promising tool to discover novel microbes and viruses. Viral metagenomes typically contain many unknown sequences. Here we describe the discovery of a previously unidentified bacteriophage present in the majority of published human faecal metagenomes, which we refer to as crAssphage. Its ~97 kbp genome is six times more abundant in publicly available metagenomes than all other known phages together; it comprises up to 90% and 22% of all reads in virus-like particle (VLP)-derived metagenomes and total community metagenomes, respectively; and it totals 1.68% of all human faecal metagenomic sequencing reads in the public databases. The majority of crAssphage-encoded proteins match no known sequences in the database, which is why it was not detected before. Using a new co-occurrence profiling approach, we predict a Bacteroides host for this phage, consistent with Bacteroides-related protein homologues and a unique carbohydrate-binding domain encoded in the phage genome.
Mya Breitbart's insight:
Not quite about Aquatic Viruses, but too cool not to share. PS - love the name!
Microbes and their viruses drive myriad processes across ecosystems ranging from oceans and soils to bioreactors and humans. Despite this importance, microbial diversity is only now being mapped at scales relevant to nature, while the viral diversity associated with any particular host remains little researched. Here we quantify host-associated viral diversity using viral-tagged metagenomics, which links viruses to specific host cells for high-throughput screening and sequencing. In a single experiment, we screened 107 Pacific Ocean viruses against a single strain of Synechococcus and found that naturally occurring cyanophage genome sequence space is statistically clustered into discrete populations. These population-based, host-linked viral ecological data suggest that, for this single host and seawater sample alone, there are at least 26 double-stranded DNA viral populations with estimated relative abundances ranging from 0.06 to 18.2%. These populations include previously cultivated cyanophage and new viral types missed by decades of isolate-based studies. Nucleotide identities of homologous genes mostly varied by less than 1% within populations, even in hypervariable genome regions, and by 42-71% between populations, which provides benchmarks for viral metagenomics and genome-based viral species definitions. Together these findings showcase a new approach to viral ecology that quantitatively links objectively defined environmental viral populations, and their genomes, to their hosts.
Mya Breitbart's insight:
Very cool method for linking marine phage with their hosts...
Viruses (including bacteriophages) are the most abundant biological entities on the planet. As such, they are thought to have a major impact on all aspects of microbial community structure and function. Despite this critical role in ecosystem processes, the study of virus/phage diversity has lagged far behind parallel studies of the bacterial and eukaryotic kingdoms, largely due to the absence of any universal phylogenetic marker. Here we review the development and use of signature genes to investigate viral diversity, as a viable strategy for data sets of specific virus groups. Genes that have been used include those encoding structural proteins, such as portal protein, major capsid protein, and tail sheath protein, auxiliary metabolism genes, such as psbA, psbB, and phoH, and several polymerase genes. These marker genes have been used in combination with PCR-based fingerprinting and/or sequencing strategies to investigate spatial, temporal, and seasonal variations and diversity in a wide range of habitats.
Mya Breitbart's insight:
Since there is no universal gene found in all viruses - these "signature genes" provide a good method for surveying subsets of viral diversity
Viruses are known as the most abundant and genetically diverse life forms on the planet, but there is still a lack of information on many of the various types that make up this group, including the phylogenetic relationships between them. Single-stranded DNA (ssDNA) viruses have previously been divided up into seven known families and are known to infect many plants and animals, and have small genomes (between 1.4 and 8.5 kb). Of these seven families, there are currently two groups of bacteriophages (Inoviridae andMicroviridae) and five that infect eukaryotes (Nanoviridae and Geminiviridae which infect plants; and Circoviridae, Parvoviridae and Anelloviridae which infect animals). The genomes of these ssDNA viruses are able to encode as few as two genes; a capsid and a replication initiator.
Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (~14 Ma old) and the most oligotrophic subsurface sediments of the world’s oceans (South Pacific Gyre (SPG)). The numbers of viruses (104–109 cm−3, counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.
Continental shelves and the waters overlying them support numerous industries as diverse as tourism and recreation, energy extraction, fisheries, transportation, and applications of marine bio-molecules (e.g., agribusiness, food processing, pharmaceuticals). Although these shelf ecosystems exhibit impacts of climate change and increased human use of resources (Halpern et al., 2012; IPCC, 2013, 2014; Melillo et al., 2014), there are currently no standardized metrics for assessing changes in ecological function in the coastal ocean. Here, we argue that it is possible to monitor vital signs of ecosystem function by focusing on the lowest levels of the ocean food web. Establishment of biodiversity, biomass, and primary productivity baselines and continuous evaluation of changes in biological resources in these economically and ecologically valuable regions requires an internationally coordinated monitoring effort that fully integrates natural, social, and economic sciences to jointly identify problems and design solutions. Such an ocean observing network is needed to protect the livelihoods of coastal communities in the context of the goals of the Future Earth program (Mooney et al., 2013) and of the Intergovernmental Platform on Biodiversity and Ecosystem Services (http://www.ipbes.net). The tools needed to initiate these assessments are available today.
In marine environments, virus-mediated lysis of host cells leads to the release of cellular carbon and nutrients and is hypothesized to be a major driver of carbon recycling on a global scale. However, efforts to characterize the effects of viruses on nutrient cycles have overlooked the geochemical potential of the virus particles themselves, particularly with respect to their phosphorus content. In this Analysis article, we use a biophysical scaling model of intact virus particles that has been validated using sequence and structural information to quantify differences in the elemental stoichiometry of marine viruses compared with their microbial hosts. By extrapolating particle-scale estimates to the ecosystem scale, we propose that, under certain circumstances, marine virus populations could make an important contribution to the reservoir and cycling of oceanic phosphorus.
The genome of CroV has a size of 744kb and encodes for 544 predicted genes. CroV derived its name from its host, a unicellular marine zooplankton Cafeteria roenbergensis (Cro) that is the major microbial grazer in the ocean. By controlling Cro's population, CroV has a great impact on the marine ecological system. CroV is covered by a protein shell (capsid) comprised of major capsid proteins (MCP) and minor capsid proteins (mCP). The capsid not only protects the genetic material of the virus, but also helps in host-virus recognition, which is the critical initial step for viral infection. This research focuses on obtaining large amount homogenous CroV MCP for future structural studies that will facilitate the understanding of the roles of MCP in the viral life cycle.
Viruses have a profound influence on the ecology and evolution of plankton, but our understanding of the composition of the aquatic viral communities is still rudimentary. This is especially true of those viruses having RNA genomes. The limited data that have been published suggest that the RNA virioplankton is dominated by viruses with positive-sense, single-stranded (+ss) genomes that have features in common with those of eukaryote-infecting viruses in the order Picornavirales (picornavirads). In this study, we investigated the diversity of the RNA virus assemblages in tropical coastal seawater samples using targeted PCR and metagenomics. Amplification of RNA-dependent RNA polymerase (RdRp) genes from fractions of a buoyant density gradient suggested that the distribution of two major subclades of the marine picornavirads was largely congruent with the distribution of total virus-like RNA, a finding consistent with their proposed dominance. Analyses of the RdRp sequences in the library revealed the presence of many diverse phylotypes, most of which were related only distantly to those of cultivated viruses. Phylogenetic analysis suggests that there were hundreds of unique picornavirad-like phylotypes in one 35-liter sample that differed from one another by at least as much as the differences among currently recognized species. Assembly of the sequences in the metagenome resulted in the reconstruction of six essentially complete viral genomes that had features similar to viruses in the families Bacillarna-, Dicistro-, and Marnaviridae. Comparison of the tropical seawater metagenomes with those from other habitats suggests that +ssRNA viruses are generally the most common types of RNA viruses in aquatic environments, but biases in library preparation remain a possible explanation for this observation.
Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five “standard” categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing.
Viruses are the most abundant biological entities in the oceans and a pervasive cause of mortality of microorganisms that drive biogeochemical cycles. Although the ecological and evolutionary effects of viruses on marine phototrophs are well recognized, little is known about their impact on ubiquitous marine lithotrophs. Here, we report 18 genome sequences of double-stranded DNA viruses that putatively infect widespread sulfur-oxidizing bacteria. Fifteen of these viral genomes contain auxiliary metabolic genes for the α and γ subunits of reverse dissimilatory sulfite reductase (rdsr). This enzyme oxidizes elemental sulfur, which is abundant in the hydrothermal plumes studied here. Our findings implicate viruses as a key agent in the sulfur cycle and as a reservoir of genetic diversity for bacterial enzymes that underpin chemosynthesis in the deep oceans.
Cyanobacteria coexist in the oceans with a wealth of phages that infect them. While numerous studies have investigated Synechococcus phages, much less data is available for Prochlorococcus phages. Furthermore, little is known about cyanophage composition. Here we examined the abundance and relative composition of cyanophages on six cyanobacterial hosts in samples collected during spring and summer from the Red Sea. Maximal abundances found on Synechococcus of 35,000 phages/ml are within ranges found previously whereas the 24,000 phages/ml found on Prochlorococcus are approximately 10-fold higher than previous findings. T7-like, T4-like and “unknown” phages were isolated on all hosts, including many T4-like phages on HL Prochlorococcus strains, whereas TIM5-like phages were found only on Synechococcus. Large differences in cyanophage abundance and composition were found for different hosts on the same sampling date as well as for the same host on different dates with few predictable patterns discerned. Host-range analyses showed that T7-like and TIM5-like phages were quite host-specific, whereas the breadth of hosts for T4-like phages was related to host type: those isolated on HL Prochlorococcus were considerably more host-specific than those on LL Prochlorococcus or Synechococcus. These host-related differences likely contribute to the complexity of host-phage interactions in the oceans.
Multiple studies have explored microbial shifts in diseased or stressed corals, however little is known about bacteriophage interactions with microbes in this context. This study characterized microbial 16S rRNA amplicons and phage metagenomes associated with Montastraea annularis corals during a concurrent white plague disease outbreak and bleaching event. Phage consortia differed between bleached and diseased tissues. Phages in the family Inoviridae were elevated in diseased or healthy tissues compared to bleached portions of diseased tissues. Microbial communities also differed between diseased and bleached corals. Bacteria in the orders Rhodobacterales and Campylobacterales were increased while Kiloniellales was decreased in diseased compared to other tissues. A network of phage-bacteria interactions was constructed of all phage strains and 11 bacterial genera that differed across health states. Phage-bacteria interactions varied in specificity: phages interacted with 1-8 bacterial hosts while bacteria interacted with up to 59 phages. Six phages were identified that interacted exclusively with Rhodobacterales and Camplyobacterales. These results suggest that phages have a role in controlling stress-associated bacteria, and that networks can be utilized to select potential phages for mitigating detrimental bacterial growth in phage therapy applications.
Mya Breitbart's insight:
Very interesting --- links between coral health, bacterial communities, and phages! Also one of the first studies to find Inoviruses in the oceans that I'm aware of.
In 2003, Acanthamoeba polyphaga mimivirus (APMV) was first described and began to impact researchers around the world, due to its structural and genetic complexity. This virus founded the family Mimiviridae. In recent years, several new giant viruses have been isolated from different environments and specimens. Giant virus research is in its initial phase and information that may arise in the coming years may change current conceptions of life, diversity and evolution. Thus, this review aims to condense the studies conducted so far about the features and peculiarities of APMV, from its discovery to its clinical relevance.
A number of bivalve species worldwide, including the Pacific oyster,Crassostrea gigas, have been affected by mass mortality events associated with herpesviruses and resulting in significant losses. A particular herpesvirus was purified from naturally infected larval Pacific oysters and its genome was entirely sequenced. This virus has been classified asOstreid herpesvirus 1 (OsHV-1) within the Malacoherpesviridae family. Since 2008, mass mortality outbreaks among C. gigas in Europe have been related to the detection of a variant of OsHV-1 called μVar.
Additional data is necessary to better describe mortality events in relation with environmental parameter fluctuations and OsHV-1 detection. For this purpose, a single batch of Pacific oyster spat was deployed in 4 different locations in the Marennes-Oleron area (France) including a pond (“claire”), a shellfish nursery and two locations in the field. Mortality rates were recorded based on regular observation and samples were collected to search and quantify OsHV-1 DNA by real time PCR. Although similar massive mortality rates were reported at 4 sites, mortality was detected earlier in the pond and in the nursery than at both field sites. This difference may be related to earlier increases of water temperature. Mass mortality was observed among oysters a few days after increases of the number of PCR positive oysters and viral DNA amounts were recorded. A initial increment in the number of PCR positive oysters was reported at both field sites during the survey in absence of significant mortality. During this period the water temperature was below 16°C.
Ed Rybicki's insight:
Clever idea: trawling for viruses with bait organisms in an aquaculture setting.
Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb–2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants.
Cafeteria roenbergensis virus (CroV) is a giant virus of the Mimiviridae family that infects the marine phagotrophic flagellate C. roenbergensis. CroV possesses a DNA genome of ~730 kilobase pairs that is predicted to encode 544 proteins. We analyzed the protein composition of purified CroV particles by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and identified 141 virion-associated CroV proteins and 60 host proteins. Data are available via ProteomeXchange with identifier PXD000993. Predicted functions could be assigned to 36% of the virion proteins, which include structural proteins as well as enzymes for transcription, DNA repair, redox reactions and protein modification. Homologs of 36 CroV virion proteins have previously been found in the virion of Acanthamoeba polyphaga mimivirus. The overlapping virion proteome of CroV and Mimivirus reveals a set of conserved virion protein functions that were presumably present in the last common ancestor of the Mimiviridae.
Ed Rybicki's insight:
I love this stuff: I am going to work VERY hard at getting money to carry on with our modest project.
This project describes the protein composition of the Cafeteria roenbergensis virus (CroV, strain BV-PW1: TaxID 693272) particle, a giant marine DNA virus that infects the heterotrophic nanoflagellate microeukaryote C. roenbergensis. CroV is a member of the Nucleo-Cytoplasmic Large DNA Virus clade and related to Acanthamoeba polyphaga mimivirus. CroV possesses a DNA genome of ~730 kilobase pairs that encodes 544 predicted proteins. We analyzed the protein composition of purified CroV particles by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified 141 virion-associated CroV proteins. Predicted functions could be assigned to 37% of these proteins, which include structural proteins as well as enzymes for transcription, DNA repair, redox reactions and protein modification. Homologs of 36 CroV virion proteins have previously been found in the virion of Acanthamoeba polyphaga mimivirus. This study shows that giant DNA virus particles contain more than one hundred viral proteins that include specific enzymatic functions.
Hypolithic microbial communities are specialized desert communities inhabiting the underside of translucent rocks. Here, we present the first study of the viral fraction of these communities isolated from the hyperarid Namib Desert. The taxonomic composition of the hypolithic viral communities was investigated and a functional assessment of the sequences determined. Phylotypic analysis showed that bacteriophages belonging to the order Caudovirales, in particular the family Siphoviridae, were most prevalent. Functional analysis and comparison with other metaviromes revealed a relatively high frequency of cell wall-degrading enzymes, ribonucleotide reductases (RNRs) and phage-associated genes. Phylogenetic analyses of terL and phoH marker genes indicated that many of the sequences were novel and distinct from known isolates, and the class distribution of the RNRs suggests this is a novel environment. The composition of the viral hypolith fraction containing many Bacillus-infecting phages was not completely consistent with Namib hypolith phylotypic surveys of the bacterial hosts, in which the cyanobacterial genus Chroococcidiopsis was found to be dominant. This could be attributed to the lacking of sequence information about hypolith viruses/bacteria in public databases or the possibility that hypolithic communities incorporate viruses from the surrounding soil.
Ed Rybicki's insight:
Not quite aquatic, but metagenomic nonetheless. A good practice ground!!
Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an 'open-eight' conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.