Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses.
ABSTRACT: Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270-14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.
Project description:The identification of relevant virus-host pairs that globally account for a large pool of carbon and nutrients in the ocean is paramount to build accurate ecological models. A previous work using single-virus genomics led to the discovery of the uncultured single-virus vSAG 37-F6, originally sorted from the Mediterranean Sea (Blanes Bay Microbial Observatory), that represents one of the most abundant dsDNA viral population in the marine surface virosphere. Here, from same sampling site, we report that a Pelagibacter single-cell contained a viral member of vSAG 37-F6 population, by means of PCR screening of sorted, genome-amplified single cells with vSAG 37-F6-specific primers and whole-genome sequencing. Furthermore, viruses from this population were also found in three other Pelagibacter single cells from the South Pacific and Atlantic oceans. These new uncultured pelagiphages were genetically different from the previously characterized pelagiphage isolates. Data showed that the uncultured vSAG 37-F6 population represents the Pelagibacter phages that inhabit the sunlit ocean better, and contains a vast unrecognized microdiversity.
Project description:Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.
Project description:Viruses are the most abundant biological entities in the oceans, and account for a significant amount of the genetic diversity of marine ecosystems. However, there is little detailed information about the biodiversity of viruses in marine environments. Rapid advances in metagenomics have enabled the identification of previously unknown marine viruses. We performed metagenomic profiling of seawater samples collected at 6 sites in Goseong Bay (South Sea, Korea) during the spring, summer, autumn, and winter of 2014. The results indicated the presence of highly diverse virus communities. The DNA libraries from samples collected during four seasons were sequenced using Illumina HiSeq 2000. The number of viral reads was 136,850 during March, 70,651 during June, 66,165 during September, and 111,778 during December. Species identification indicated that Pelagibacter phage HTVC010P, Ostreococcus lucimarinus OIV5 and OIV1, and Roseobacter phage SIO1 were the most common species in all samples. For viruses with at least 10 reads, there were 204 species during March, 189 during June, 170 during September, and 173 during December. Analysis of virus families indicated that the Myoviridae was the most common during all four seasons, and viruses in the Polyomaviridae were only present during March. Viruses in the Iridoviridae were only present during three seasons. Additionally, viruses in the Iridoviridae, Herpesviridae, and Poxviridae, which may affect fish and marine animals, appeared during different seasons. These results suggest that seasonal changes in temperature contribute to the dynamic structure of the viral community in the study area. The information presented here will be useful for comparative analyses with other marine viral communities.
Project description:BACKGROUND:Viruses are the most abundant biological entities on earth and play import roles in marine biogeochemical cycles. Here, viral communities in the surface water of the East China Sea (ECS) were collected from three representative regions of Yangshan Harbor (YSH), Gouqi Island (GQI), and the Yangtze River Estuary (YRE) and explored primarily through epifluorescence microscopy (EM), transmission electron microscopy (TEM), and metagenomics analysis. RESULTS:The virus-like particles (VLPs) in the surface water of the ECS were measured to be 106 to 107 VLPs/ml. Most of the isolated viral particles possessed a head-and-tail structure, but VLPs with unique morphotypes that had never before been observed in the realm of viruses were also found. The sequences related to known viruses in GenBank accounted for 21.1-22.8% of the viromic datasets from YSH, GQI, and YRE. In total, 1029 viral species were identified in the surface waters of the ECS. Among them, tailed phages turn out to make up the majority of viral communities, however a small number of Phycodnaviridae or Mimiviridae related sequences were also detected. The diversity of viruses did not appear to be a big difference among these three aquatic environments but their relative abundance was geographically variable. For example, the Pelagibacter phage HTVC010P accounted for 50.4% of the identified viral species in GQI, but only 9.1% in YSH and 11.7% in YRE. Sequences, almost identical to those of uncultured marine thaumarchaeal dsDNA viruses and magroviruses that infect Marine Group II Euryarchaeota, were confidently detected in the ECS viromes. The predominant classes of virome ORFs with functional annotations that were found were those involved in viral biogenesis. Virus-host connections, inferred from CRISPR spacer-protospacer mapping, implied newly discovered infection relationships in response to arms race between them. CONCLUSIONS:Together, both identified viruses and unknown viral assemblages observed in this study were indicative of the complex viral community composition found in the ECS. This finding fills a major gap in the dark world of oceanic viruses of China and additionally contributes to the better understanding of global marine viral diversity, composition, and distribution.
Project description:Herpesvirus saimiri C488 transforms human T lymphocytes to stable growth in culture. The growth-transformed human T cells harbor the viral genome in a nonintegrated episomal form without production of virus particles. In these cells, virus gene expression was previously found to be confined to the transforming genes stpC and tip. In order to analyze virus gene expression in more detail, we applied a subtractive hybridization technique and compared stimulated virus-transformed cells with uninfected parental T cells of the same donor. A number of known T-cell activation genes were isolated. Viral stpC/tip cDNAs were enriched after subtraction. In addition, the viral immediate-early, superantigen-homologous gene ie14/vsag was represented by numerous cDNA clones that comprised the entire spliced transcript. Whereas a weak basal expression of ie14/vsag was detected by reverse transcription-PCR only, the phorbol ester-induced transcripts were readily shown by Northern blotting. ie14/vsag, which before had been classified as a major immediate-early gene of herpesvirus saimiri, is localized within a highly conserved region with extensive homologies to the cellular genome. Mutant viruses without the ie14/vsag gene are replication competent and fully capable of transforming human and marmoset T cells. Since ie14/vsag is transiently expressed after stimulation, it may increase T-cell proliferation in an activation-dependent and superantigen-like but apparently Vbeta-independent way.
Project description:Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000?copies?ml(-1) and were considered 'high abundance', whereas the other two Prasinovirus-related genes peaked at abundances <1000?copies?ml(-1) and were considered 'low abundance'. Of the genes related to chloroviruses, one peaked at ca 1600?copies?ml(-1), whereas the other reached only ca 300?copies?ml(-1). Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100-1000?copies?ml(-1) while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100?copies?ml(-1). Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.
Project description:Virus particles are highly abundant in seawater and, on average, outnumber microbial cells approximately 10-fold at the surface and 16-fold in deeper waters; yet, this relationship varies across environments. Here, we examine the influence of a suite of environmental variables, including nutrient concentrations, salinity and temperature, on the relationship between the abundances of viruses and prokaryotes over a broad range of spatial and temporal scales, including along a track from the Northwest Atlantic to the Northeast Pacific via the Arctic Ocean, and in the coastal waters of British Columbia, Canada. Models of varying complexity were tested and compared for best fit with the Akaike Information Criterion, and revealed that nitrogen and phosphorus concentrations, as well as prokaryote abundances, either individually or combined, had significant effects on viral abundances in all but hypoxic environments, which were only explained by a combination of physical and chemical factors. Nonetheless, multivariate models of environmental variables showed high explanatory power, matching or surpassing that of prokaryote abundance alone. Incorporating both environmental variables and prokaryote abundances into multivariate models significantly improved the explanatory power of the models, except in hypoxic environments. These findings demonstrate that environmental factors could be as important as, or even more important than, prokaryote abundance in describing viral abundance across wide-ranging marine environments.
Project description:Viruses play important roles in marine surface ecosystems, but little is known about viral ecology and virus-mediated processes in deep-sea hydrothermal microbial communities. In this study, we examined virus-like particle (VLP) abundances in planktonic and attached microbial communities, which occur in physical and chemical gradients in both deep and shallow submarine hydrothermal environments (mixing waters between hydrothermal fluids and ambient seawater and dense microbial communities attached to chimney surface areas or macrofaunal bodies and colonies). We found that viruses were widely distributed in a variety of hydrothermal microbial habitats, with the exception of the interior parts of hydrothermal chimney structures. The VLP abundance and VLP-to-prokaryote ratio (VPR) in the planktonic habitats increased as the ratio of hydrothermal fluid to mixing water increased. On the other hand, the VLP abundance in attached microbial communities was significantly and positively correlated with the whole prokaryotic abundance; however, the VPRs were always much lower than those for the surrounding hydrothermal waters. This is the first report to show VLP abundance in the attached microbial communities of submarine hydrothermal environments, which presented VPR values significantly lower than those in planktonic microbial communities reported before. These results suggested that viral lifestyles (e.g., lysogenic prevalence) and virus interactions with prokaryotes are significantly different among the planktonic and attached microbial communities that are developing in the submarine hydrothermal environments.
Project description:Viruses are an abundant and active component of marine sediments and play a significant role in microbial ecology and biogeochemical cycling at local and global scales. To obtain a better understanding of the ecological characteristics of the viriobenthos, the abundance and morphology of viruses and the diversity and community structure of T4-type phages were systematically investigated in the surface sediments of the subtropical Pearl River Estuary (PRE). Viral abundances ranged from 4.49 × 108 to 11.7 × 108 viruses/g and prokaryotic abundances ranged from 2.63 × 108 to 9.55 × 108 cells/g, and both decreased from freshwater to saltwater. Diverse viral morphotypes, including tailed, spherical, filamentous, and rod-shaped viruses, were observed using transmission electron microscopy. Analysis of the major capsid gene (g23) indicated that the sediment T4-type phages were highly diverse and, similar to the trend in viral abundances, their diversity decreased as the salinity increased. Phylogenetic analysis suggested that most of the g23 operational taxonomic units were affiliated with marine, paddy soil, and lake groups. The T4-type phage communities in freshwater and saltwater sediments showed obvious differences, which were related to changes in the Pearl River discharge. The results of this study demonstrated both allochthonous and autochthonous sources of the viral community in the PRE sediments and the movement of certain T4-type viral groups between the freshwater and saline water biomes.
Project description:Fonsibacter (LD12 subclade) is among the most abundant bacterioplankton in freshwater ecosystems. These bacteria belong to the order Pelagibacterales (SAR11) and are related to Pelagibacter (marine SAR11), which dominates many marine habitats. Although a few Pelagibacter phage (Pelagiphage) have been described, no phage that infect Fonsibacter have been reported. In this study, we describe two groups of Podoviridae phage that infect Fonsibacter A complete Fonsibacter genome containing a prophage was reconstructed from metagenomic data. A circularized and complete genome related to the prophage, referred to as uv-Fonsiphage-EPL (lysogenic strategy), shows high similarity to marine Pelagiphage HTVC025P. Additionally, we reconstructed three complete genomes and one draft genome of phage related to marine Pelagiphage HTVC010P and predicted a lytic strategy. The similarity in codon usage and cooccurrence patterns of HTVC010P-related phage and Fonsibacter suggested that these phage infect Fonsibacter Similar phage were detected in Lake Mendota, Wisconsin, where Fonsibacter is also present. A search of related phage revealed the worldwide distribution of some genotypes in freshwater ecosystems, suggesting their substantial role in shaping indigenous microbial assemblages and influence on biogeochemical cycling. However, the uv-Fonsiphage-EPL and one group of HTVC010P-related phage have a more limited distribution in freshwater ecosystems. Overall, the findings provide insights into the genomic features of phage that infect Fonsibacter and expand understanding of the ecology and evolution of these important bacteria.IMPORTANCE Fonsibacter represents a significant microbial group of freshwater ecosystems. Although the genomic and metabolic features of these bacteria have been well studied, no phage infecting them has been reported. In this study, we reconstructed complete genomes of Fonsibacter and infecting phage and revealed their close relatedness to the phage infecting marine SAR11 members. Also, we illustrated that phage that infect Fonsibacter are widely distributed in freshwater habitats. In summary, the results contribute new insights into the ecology and evolution of Fonsibacter and phage.