Viral Community Structure Research Articles

Challenges of studying viral aerosol metagenomics and communities in comparison with bacterial and fungal aerosols.

Despite the obvious importance of viral transmission and ecology to medicine, epidemiology, ecology, agriculture, and microbiology, the study of viral bioaerosols and community structure has remained a vastly underexplored area, due to both unresolved technical challenges and unrecognized importance. High-throughput, culture-independent techniques such as viral metagenomics are beginning to revolutionize the study of viral ecology. With recent developments in viral metagenomics, characterization of viral bioaerosol communities provides an opportunity for high-impact future research. However, there remain significant challenges for the study of viral bioaerosols compared with viruses in other matrices, such as water, the human gut, and soil. Collecting enough biomass is essential for successful metagenomic analysis, but this is a challenge with viral bioaerosols. Herein, we provide a perspective on the importance of studying viral bioaerosols, the challenges of studying viral community structure, and the potential opportunities for improvements in methods to study viruses in indoor and outdoor air.

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

BackgroundMetagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome.ResultsTo face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks.ConclusionsThe new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface.

Metagenomics-based analysis of viral communities in dairy lagoon wastewater

Microbial populations, especially those of viruses, are poorly studied in dairy wastewater treatment operations. Here we report signature nucleic acid metagenomic sequences obtained by pyrosequencing viromes of virus-like particles that were extracted from two dairy waste treatment lagoons. The lagoons are operated in series, with Lagoon I being used as the primary stage and Lagoon II as the secondary stage of wastewater treatment. An average of 2000 sequences was obtained from each lagoon. More than 300 signatures from each lagoon matched sequences in the virus database of the National Center for Biotechnology Information (NCBI). We utilized a bioinformatics approach and transmission electron microscopy (TEM) to characterize the viral diversity and presence of potential viral pathogens within the lagoons. Our results showed differences in viral community compositions between Lagoon I and Lagoon II, suggesting that the viral community changes significantly in the transition of water between the two lagoons. Furthermore, the diverse viral community in the lagoon samples contained signature sequences of a variety of bacterial, plant, and animal viruses. Bacteriophage sequences dominated the viral community metagenomes in both lagoons. Ultimately these results can be used to identify viral bioindicators to rapidly assess wastewater treatment quality and the potential impacts of dairy operations on watersheds. Our viral metagenomic sequences have been submitted to GenBank (GPID 65805) and can provide insight into the composition and structure of viral communities within wastewaters of dairy lagoon systems.

Dynamics of various viral groups infecting autotrophic plankton in Lake Geneva

Viral community structure and dynamics were investigated for the first time in surface waters (0–20 m) of Lake Geneva over a 5-month period between July and November 2011. Abundances of autotrophic picoplankton, heterotrophic bacteria and virus-like particles determined using flow cytometry revealed their predominance during the summer months followed by a slight decrease in fall. Two groups of viruses could be discriminated, referred to as virus-like particles (VLP) group 1 and 2. The abundance of VLP1 correlated significantly with the bacterial abundance, while that of VLP2 correlated with both chlorophyll a and picocyanobacterial abundance suggesting a tight coupling between these viral groups and bacteria or phytoplankton. The abundance of cyanomyoviruses and cyanopodoviruses varied between 7.3 × 102 ml−1 (July) to 1.2 × 104 ml−1 (November) and 5.8 × 103 ml−1 (July) to 2.2 × 104 ml−1 (September), respectively. The abundance of the picocyanobacterial hosts was in concurrence with that of the cyanophages, being higher in late summer. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of viral signature genes such as g20, g23, mcp, polB, psbA and psbD revealed a relatively high richness within these genes with their diversity increasing towards the summer months. The diversity of psbD was found to be particularly high and correlated with picocyanobacterial abundance suggesting that cyanophages may be directly responsible for a significant proportion of carbon fixation in Lake Geneva.

Dynamics of various viral groups infecting autotrophic plankton in Lake Geneva

Viral community structure and dynamics were investigated for the first time in surface waters (0–20 m) of Lake Geneva over a 5-month period between July and November 2011. Abundances of autotrophic picoplankton, heterotrophic bacteria and virus-like particles determined using flow cytometry revealed their predominance during the summer months followed by a slight decrease in fall. Two groups of viruses could be discriminated, referred to as virus-like particles (VLP) group 1 and 2. The abundance of VLP1 correlated significantly with the bacterial abundance, while that of VLP2 correlated with both chlorophyll a and picocyanobacterial abundance suggesting a tight coupling between these viral groups and bacteria or phytoplankton. The abundance of cyanomyoviruses and cyanopodoviruses varied between 7.3 × 102 ml−1 (July) to 1.2 × 104 ml−1 (November) and 5.8 × 103 ml−1 (July) to 2.2 × 104 ml−1 (September), respectively. The abundance of the picocyanobacterial hosts was in concurrence with that of the cyanophages, being higher in late summer. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of viral signature genes such as g20, g23, mcp, polB, psbA and psbD revealed a relatively high richness within these genes with their diversity increasing towards the summer months. The diversity of psbD was found to be particularly high and correlated with picocyanobacterial abundance suggesting that cyanophages may be directly responsible for a significant proportion of carbon fixation in Lake Geneva.

A local upwelling controls viral and microbial community structure in South Australian continental shelf waters

Despite the increasing awareness of the role of viruses and heterotrophic bacteria in microbial dynamics and biogeochemical cycles, there is still a critical lack of information on their community composition and dynamics, especially in relation to upwellings. We investigated, within surface waters and the Deep Chlorophyll Max, the community composition and dynamics of flow cytometrically defined sub-populations of heterotrophic bacteria and virus-like particles in nearby water masses that were affected and unaffected by a localised wind-driven coastal upwelling. In contrast to previous studies we uniquely identified a 4-fold increase in total viral abundance and a decrease in bacterial abundance, from upwelled to offshore waters. Individual viral sub-populations were seen to correlate significantly to both bacterial populations and chlorophyll a, suggesting the possibility of individual viral populations infecting multiple host species rather than the often assumed single host species. The percentage of HDNA bacteria was high (84.3–93.4%) within upwelled waters, in accordance with the highest recorded values within an upwelling system, and decreased down to 35.5–42.6% away from the upwelling. Additionally, changes in the community composition of individual bacterial sub-populations suggest individual populations might be better adapted to distinct environments. We suggest that each flow cytometrically defined bacterial population may possess its own environmental niche where favourable conditions for that population result in an increase in abundance, cellular activity and productivity.

In situ structuring of virioplankton through ­bacterial exoenzymatic activity: interaction with phytoplankton

The abundance and composition of microbial communities were investigated during 2007 in Marennes Oleron Bay (France) in order to characterize the biological relationships governing the planktonic food web. Compared to the results of previous years, there was a lower abundance and a lower magnitude of variations in autotrophic and heterotrophic microbial entities in 2007. Using a Spearman rank correlation analysis, a significant link for biological abundance was only noted between bacteria and ciliates. However, an interesting relationship was highlighted between chlorophyll a and bacterial aminopeptidase and β-glucosidase exoenzymatic activities. Two charac- teristic periods of virus-bacteria uncoupling were observed in spring and autumn 2007. Using multi- variate analysis, the clustering of monthly viral community structures (defined by RAPD-fingerprint- ing) was related to bacterial proteolysis activity and secondarily to flagellates. Spring and autumn were characterized by phytoplanktonic blooms of large (>10 µm) and small cells (<10 µm), respec- tively, during the virus-bacteria uncoupling phases. High predation by flagellates in the autumn, during a period of microbial food web activity, involved the highest viral fingerprint richness, infer- ring a top-down control in driving the number of virus-host systems. We discuss the proteolysis level as a descriptor in structuring viral assemblages and argue that changes in the quantity and quality of DOM may bridge phytoplankton to bacteria and reflect changes in phytoplankton composition.

A non-negative matrix factorization framework for identifying modular patterns in metagenomic profile data

Metagenomic studies sequence DNA directly from environmental samples to explore the structure and function of complex microbial and viral communities. Individual, short pieces of sequenced DNA ("reads") are classified into (putative) taxonomic or metabolic groups which are analyzed for patterns across samples. Analysis of such read matrices is at the core of using metagenomic data to make inferences about ecosystem structure and function. Non-negative matrix factorization (NMF) is a numerical technique for approximating high-dimensional data points as positive linear combinations of positive components. It is thus well suited to interpretation of observed samples as combinations of different components. We develop, test and apply an NMF-based framework to analyze metagenomic read matrices. In particular, we introduce a method for choosing NMF degree in the presence of overlap, and apply spectral-reordering techniques to NMF-based similarity matrices to aid visualization. We show that our method can robustly identify the appropriate degree and disentangle overlapping contributions using synthetic data sets. We then examine and discuss the NMF decomposition of a metabolic profile matrix extracted from 39 publicly available metagenomic samples, and identify canonical sample types, including one associated with coral ecosystems, one associated with highly saline ecosystems and others. We also identify specific associations between pathways and canonical environments, and explore how alternative choices of decompositions facilitate analysis of read matrices at a finer scale.

Abundance, Diversity, and Dynamics of Viruses on Microorganisms in Activated Sludge Processes

We examined the abundance of viruses on microorganisms in activated sludge and the dynamics of their community structure. Direct counting with epifluorescence microscopy and pulsed-field gel electrophoresis (PFGE) were applied to 20 samples from 14 full-scale wastewater treatment plants (wwtps) treating municipal, industrial, or animal wastewater. Furthermore, to observe the dynamics of viral community structure over time, a laboratory-scale sequencing batch reactor was operated for 58 days. The concentrations of virus particles in the wwtps, as quantified by epifluorescence microscopy, ranged from 4.2 x 10(7) to 3.0 x 10(9) mL-1. PFGE, improved by the introduction of a higher concentration of Tris-EDTA buffer in the DNA extraction step, was successfully used to profile DNA viruses in the activated sludge. Most of the samples from different wwtps commonly had bands in the 40-70 kb range. In the monitoring of viral DNA size distribution in the laboratory-scale reactor, some bands were observed stably throughout the experimental period, some emerged during the operation, and others disappeared. Rapid emergence and disappearance of two intense bands within 6 days was observed. Our data suggest that viruses--especially those associated with microorganisms--are abundant and show dynamic behavior in activated sludge.

Structure of Virioplankton in the Charente Estuary (France): Transmission Electron Microscopy versus Pulsed Field Gel Electrophoresis

Changes in the composition of viral communities were investigated along a salinity gradient and at different times by means of transmission electron microscopy (TEM) and pulsed field gel electrophoresis (PFGE). Samples were collected in fresh (Charente River), estuarine (Charente Estuary), and coastal (Pertuis d'Antioche, French Atlantic coast) waters. Both methods revealed similar patterns in viral community structure with a dominance of small viral particles (capsid and genome size). Viruses with a head size below 65 nm made up 71 +/- 5% of total virus-like particles, and virus-like genomes (VLG) below 100 kb accounted for 89 +/- 9% of total VLG. Despite this apparent stability of virioplankton composition over spatial scale (salinity gradient), the occurrence of large viruses (capsid and genome size) in estuarine and seawater samples indicated the presence of viral populations specific to a geographical location. Temporal changes in the structure (capsid and genome size) of viral communities were more pronounced than those reported at the spatial scale. From January to May 2003, seasonal changes in viral abundance and bacterial production occurred concomitantly with an increase in viral genomic diversity (richness), suggesting that virioplankton composition was strongly linked to changes in microbial activity and/or in the structure of the host communities. Although PFGE and TEM yielded complementary results in the description of virioplankton structures, it seems that the use of PFGE alone should be enough for the monitoring of community changes.

Viral metagenomics

Viruses, most of which infect microorganisms, are the most abundant biological entities on the planet. Identifying and measuring the community dynamics of viruses in the environment is complicated because less than one percent of microbial hosts have been cultivated. Also, there is no single gene that is common to all viral genomes, so total uncultured viral diversity cannot be monitored using approaches analogous to ribosomal DNA profiling. Metagenomic analyses of uncultured viral communities circumvent these limitations and can provide insights into the composition and structure of environmental viral communities.

PHACCS, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information

BackgroundPhages, viruses that infect prokaryotes, are the most abundant microbes in the world. A major limitation to studying these viruses is the difficulty of cultivating the appropriate prokaryotic hosts. One way around this limitation is to directly clone and sequence shotgun libraries of uncultured viral communities (i.e., metagenomic analyses). PHACCS , Phage Communities from Contig Spectrum, is an online bioinformatic tool to assess the biodiversity of uncultured viral communities. PHACCS uses the contig spectrum from shotgun DNA sequence assemblies to mathematically model the structure of viral communities and make predictions about diversity.ResultsPHACCS builds models of possible community structure using a modified Lander-Waterman algorithm to predict the underlying contig spectrum. PHACCS finds the most appropriate structure model by optimizing the model parameters until the predicted contig spectrum is as close as possible to the experimental one. This model is the basis for making estimates of uncultured viral community richness, evenness, diversity index and abundance of the most abundant genotype.ConclusionPHACCS analysis of four different environmental phage communities suggests that the power law is an important rank-abundance form to describe uncultured viral community structure. The estimates support the fact that the four phage communities were extremely diverse and that phage community biodiversity and structure may be correlated with that of their hosts.

Virioplankton community structure along a salinity gradient in a solar saltern.

The virioplankton community structure along a salinity gradient from near seawater (40 per thousand ) to saturated sodium chloride brine (370 per thousand ) in a solar saltern was investigated by pulsed-field gel electrophoresis. Viral populations with genome sizes varying from 10 kb to 533 kb were detected. The viral community structure changed along the salinity gradient. Cluster analysis of the viral genome-banding pattern resulted in two main clusters. The virioplankton diversity within the samples with salinity from 40 per thousand to 150 per thousand was on the same cluster of a cladogram. The other group consisted of virioplankton from samples with salinity above 220 per thousand. The virioplankton diversity in the different samples was calculated using the Shannon index. The diversity index demonstrated an increase in diversity in the samples along the gradient from 40 per thousand to 150 per thousand salinity, followed by a decrease in the diversity index along the rest of the salinity gradient. These results demonstrate how viral diversity changes from habitats that are considered one of the most common (seawater) to habitats that are extreme in salt concentrations (saturated sodium brine). The diversity index was highest in the environments that lie in between the most extreme and the most common.

Population dynamics of chesapeake bay virioplankton: total-community analysis by pulsed-field gel electrophoresis

Recognition of viruses as the most abundant component of aquatic microbial communities has stimulated investigations of the impact of viruses on bacterio- and phytoplankton host communities. From results of field studies to date, it is concluded that in most aquatic environments, a reduction in the number of bacteria on a daily basis is caused by viral infection. However, the modest amount of in situ virus-mediated mortality may be less significant than viral infection serving to maintain clonal diversity in the host communities directly, through gene transmission (i.e., transduction), and indirectly, by elimination of numerically dominant host species. If the latter mechanism for controlling community diversity prevails, then the overall structure of aquatic viral communities would be expected to change as well over short seasonal and spatial scales. To determine whether this occurs, pulsed-field gel electrophoresis (PFGE) was used to monitor the population dynamics of Chesapeake Bay virioplankton for an annual cycle (1 year). Virioplankton in water samples collected at six stations along a transect running the length of the bay were concentrated 100-fold by ultrafiltration. Viruses were further concentrated by ultracentrifugation, and the concentrated samples were embedded in agarose. PFGE analysis of virus DNA in the agarose plugs yielded several distinct bands, ranging from 50 to 300 kb. Principal-component and cluster analyses of the virus PFGE fingerprints indicated that changes in virioplankton community structure were correlated with time, geographical location, and extent of water column stratification. From the results of this study, it is concluded that, based on the dynamic nature of the Chesapeake Bay virioplankton community structure, the clonal diversity of bacterio- and phytoplankton host communities is an important component of the virus community.