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An enteric virus surrogate and reliable domestic wastewater tracer is needed to manage microbial quality of food and water as (waste)water reuse becomes more prevalent in response to population growth, urbanization, and climate change. Pepper mild mottle virus (PMMoV), a plant pathogen found at high concentrations in domestic wastewater, is a promising surrogate for enteric viruses that has been incorporated into over 29 investigations of water- and food-related microbial quality and technology around the world. This review consolidates the available literature from across disciplines to provide guidance on the utility of PMMoV as either an enteric virus surrogate and/or domestic wastewater marker in various situations. Synthesis of the available research supports PMMoV as a useful enteric virus process indicator since its high concentrations in source water allow for identifying the extent of virus log-reductions in field, pilot, and full-scale (waste)water treatment systems. PMMoV reduction levels during many forms of wastewater treatment were less than or equal to the reduction of other viruses, suggesting this virus can serve as an enteric virus surrogate when evaluating new treatment technologies. PMMoV excels as an index virus for enteric viruses in environmental waters exposed to untreated domestic wastewater because it was detected more frequently and in higher concentrations than other human viruses in groundwater (72.2%) and surface waters (freshwater, 94.5% and coastal, 72.2%), with pathogen co-detection rates as high as 72.3%. Additionally, PMMoV is an important microbial source tracking marker, most appropriately associated with untreated domestic wastewater, where its pooled-specificity is 90% and pooled-sensitivity is 100%, as opposed to human feces where its pooled-sensitivity is only 11.3%. A limited number of studies have also suggested that PMMoV may be a useful index virus for enteric viruses in monitoring the microbial quality of fresh produce and shellfish, but further research is needed on these topics. Finally, future work is needed to fill in knowledge gaps regarding PMMoV’s global specificity and sensitivity.

Viruses numerically dominate our oceans; however, we have only just begun to document the diversity, host range and infection dynamics of marine viruses, as well as the subsequent effects of infection on both host cell metabolism and oceanic biogeochemistry. Bacteriophages (that is, phages: viruses that infect bacteria) are highly abundant and are known to play critical roles in bacterial mortality, biogeochemical cycling and horizontal gene transfer. This Review Article summarizes current knowledge of marine viral ecology and highlights the importance of phage particles to the dissolved organic matter pool, as well as the complex interactions between phages and their bacterial hosts. We emphasize the newly recognized roles of phages as puppet masters of their bacterial hosts, where phages are capable of altering the metabolism of infected bacteria through the expression of auxiliary metabolic genes and the redirection of host gene expression patterns. Finally, we propose the 'royal family model' as a hypothesis to describe successional patterns of bacteria and phages over time in marine systems, where despite high richness and significant seasonal differences, only a small number of phages appear to continually dominate a given marine ecosystem. Although further testing is required, this model provides a framework for assessing the specificity and ecological consequences of phage-host dynamics.

Diverse bacterial and fungal communities inhabit human-occupied buildings and circulate in indoor air; however, viral diversity in these man-made environments remains largely unknown. Here we investigated DNA and RNA viruses circulating in the air of 12 university dormitory rooms by analyzing dust accumulated over a one-year period on heating, ventilation, and air conditioning (HVAC) filters. A metagenomic sequencing approach was used to determine the identity and diversity of viral particles extracted from the HVAC filters. We detected a broad diversity of viruses associated with a range of hosts, including animals, arthropods, bacteria, fungi, humans, plants, and protists, suggesting that disparate organisms can contribute to indoor airborne viral communities. Viral community composition and the distribution of human-infecting papillomaviruses and polyomaviruses were distinct in the different dormitory rooms, indicating that airborne viral communities are variable in human-occupied spaces and appear to reflect differential rates of viral shedding from room occupants. This work significantly expands the known airborne viral diversity found indoors, enabling the design of sensitive and quantitative assays to further investigate specific viruses of interest and providing new insight into the likely sources of viruses found in indoor air.

The identification of host-specific bacterial and viral communities associated with diverse animals has led to the concept of the metaorganism, which defines the animal and all of its associated microbes as a single unit. Here we sequence the viruses found in the gut (i.e., the gut virome) of the marine invertebrate model system, Ciona intestinalis subtype A, in samples collected one year apart. We present evidence for a host-associated virome that is distinct from the surrounding seawater and contains some temporally-stable members. Comparison of gut tissues before and after starvation in virus-free water enabled the differentiation between the Ciona-specific virome and transient viral communities associated with dietary sources. The Ciona gut viromes were dominated by double-stranded DNA tailed phages (Order Caudovirales) and sequence assembly yielded a number of complete circular phage genomes, most of which were highly divergent from known genomes. Unique viral communities were found in distinct gut niches (stomach, midgut and hindgut), paralleling the compartmentalization of bacterial communities. Additionally, integrase and excisionase genes, including many that are similar to prophage sequences within the genomes of bacterial genera belonging to the Ciona core microbiome, were prevalent in the viromes, indicating the active induction of prophages within the gut ecosystem. Knowledge of the gut virome of this model organism lays the foundation for studying the interactions between viruses, bacteria, and host immunity.

Domestic wastewater pollution in environmental waters or water reuse supplies represents a threat to public health because of high concentrations of diverse pathogens associated with human excreta [1]. Since it is difficult to directly measure waterborne pathogens of concern, microbial water quality monitoring efforts often use surrogates or indicator organisms that are easily detected and whose presence reflects pathogen persistence [2]. Here, we describe an unconventional viral indicator of wastewater pollution, pepper mild mottle virus (PMMoV), a plant pathogen that was first proposed as a water quality indicator in 2009 [3] and promises to improve microbial water quality management worldwide [4].

Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015–2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies., Increasingly, eDNA is being used to infer ecological interactions. Here the authors sample eDNA over 18 months in a marine environment and use co-occurrence network analyses to infer potential interactions among organisms from microbes to mammals, testing how they change over time in response to oceanographic factors.

While single-stranded DNA (ssDNA) was once thought to be a relatively rare genomic architecture for viruses, modern metagenomics sequencing has revealed circular ssDNA viruses in most environments and in association with diverse hosts. In particular, circular ssDNA viruses encoding a homologous replication-associated protein (Rep) have been identified in the majority of eukaryotic supergroups, generating interest in the ecological effects and evolutionary history of circular Rep-encoding ssDNA viruses (CRESS DNA) viruses. This review surveys the explosion of sequence diversity and expansion of eukaryotic CRESS DNA taxonomic groups over the last decade, highlights similarities between the well-studied geminiviruses and circoviruses with newly identified groups known only through their genome sequences, discusses the ecology and evolution of eukaryotic CRESS DNA viruses, and speculates on future research horizons.

While single-stranded DNA (ssDNA) was once thought to be a relatively rare genomic architecture for viruses, modern metagenomics sequencing has revealed circular ssDNA viruses in most environments and in association with diverse hosts. In particular, circular ssDNA viruses encoding a homologous replication-associated protein (Rep) have been identified in the majority of eukaryotic supergroups, generating interest in the ecological effects and evolutionary history of circular Rep-encoding ssDNA viruses (CRESS DNA) viruses. This review surveys the explosion of sequence diversity and expansion of eukaryotic CRESS DNA taxonomic groups over the last decade, highlights similarities between the well-studied geminiviruses and circoviruses with newly identified groups known only through their genome sequences, discusses the ecology and evolution of eukaryotic CRESS DNA viruses, and speculates on future research horizons.

Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (

Despite the ecological importance of deep-sea cephalopods, little is known about their genetic diversity or population dynamics. The cephalopod species Cranchia scabra, Pyroteuthis margaritifera, and Vampyroteuthis infernalis are commonly collected in midwater samples from both the Gulf of Mexico and northwestern Atlantic Ocean but, despite their common appearance in trawls and important roles in marine food webs, no genetic studies of population connectivity exist for these species. Here, Sanger sequencing of three conserved genetic loci and ddRADseq techniques were used to examine population genetic dynamics in these deep-sea species. Genetic diversity is lowest in C. scabra, which appears to be in a population growth stage, and highest in V. infernalis. Population structure was unique to V. infernalis but does not appear to be the result of ocean-basin vicariance, thus possible alternative explanations are explored, specifically environmental variation in dissolved oxygen. The genetic connectivity between these geographically disparate sites suggests these three cephalopod species could be resilient to localized environmental disturbances in the Gulf of Mexico.

We report a second case of pigeon paramyxovirus type 1 infection, which has pigeons and doves as reservoir, causing fatal respiratory disease in a human patient under immunosuppressive therapy. We recommend testing for this virus in respiratory disease cases where common respiratory pathogens cannot be identified., The characteristics and risk factors of pigeon paramyxovirus type 1 (PPMV-1) infection in humans are poorly known. We performed virological, pathological, and epidemiological analyses of a Dutch case, and compared the results with those of a US case. Both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. Both virus isolates clustered with PPMV-1, which has pigeons and doves as reservoir. Experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. Both patients were likely infected by contact with infected pigeons or doves. Given the large populations of feral pigeons with PPMV-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human PPMV-1 infections may increase. We recommend testing for avian paramyxovirus type 1, including PPMV-1, in respiratory disease cases where common respiratory pathogens cannot be identified.

Phages (viruses that infect bacteria) play important roles in the gut ecosystem through infection of bacterial hosts, yet the gut virome remains poorly characterized. Mammalian gut viromes are dominated by double-stranded DNA (dsDNA) phages belonging to the order Caudovirales and single-stranded DNA (ssDNA) phages belonging to the family Microviridae. Since the relative proportion of each of these phage groups appears to correlate with age and health status in humans, it is critical to understand both ssDNA and dsDNA phages in the gut. Building upon prior research describing dsDNA viruses in the gut of Ciona robusta, a marine invertebrate model system used to study gut microbial interactions, this study investigated ssDNA phages found in the Ciona gut. We identified 258 Microviridae genomes, which were dominated by novel members of the Gokushovirinae subfamily, but also represented several proposed phylogenetic groups (Alpavirinae, Aravirinae, Group D, Parabacteroides prophages, and Peque&ntilde, ovirus) and a novel group. Comparative analyses between Ciona specimens with full and cleared guts, as well as the surrounding water, indicated that Ciona retains a distinct and highly diverse community of ssDNA phages. This study significantly expands the known diversity within the Microviridae family and demonstrates the promise of Ciona as a model system for investigating their role in animal health.

Phage Cr39582 was induced by mitomycin C from Pseudoalteromonas sp. strain Cr6751, isolated from a marine invertebrate gut. Pseudoalteromonas phage Cr39582 has 85% pairwise nucleotide identity with phage PM2 but lacks sequence homology in the spike protein. This report supports previous bioinformatic identification of corticoviral sequences within aquatic bacterial genomes.

Here, we describe four novel circular single-stranded DNA viruses discovered in fungus-farming termites ( Odontotermes sp.). The viruses, named termite-associated circular virus 1 (TaCV-1) through TaCV-4, are most similar to members of the family Genomoviridae and were widely detected in African termite mounds.

This protocol is aimed at amplifying the 16S rRNA hypervariable region 4 (16S V4) in prokaryotes. The primers (515fBand 806rB, 926R) used in this protocol are based on the primer utilized in Apprill et al 2015 and the Earth Microbiome Project (EMP). This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office. CitationApprill, A., McNally, S., Parsons, R. & Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat Microb Ecol 75, 129–137 (2015). doi:10.3354/ame01753 Earth Microbiom Projecthttp://press.igsb.anl.gov/earthmicrobiome/protocols-and-standards/16s/

Viral metagenomic analysis of heating, ventilation, and air conditioning (HVAC) filters recovered the near-complete genome sequence of a novel virus, named HVAC-associated R NA v irus 1 (HVAC-RV1). The HVAC-RV1 genome is most similar to those of picorna-like viruses identified in arthropods but encodes a small domain observed only in negative-sense single-stranded RNA viruses.

This protocol is aimed at amplifying the cytochrome c oxidase subunit I (COI) mitochondrial gene in eukaryotes. The primers (forward: mlCOIintF, reverse: HCO2198) utilized in this protocol are based on the primers utilized in Leray et al. 2013 (forward) and Folmer et al. 1994 (reverse). Amplicons generated using this protocol can then be sequenced using the Illumina platform. Primers used: Fluidigm CS1+mlCOIinfF Fluidigm CS2+HCO2198 Secondary COI PCR primers PE1-BC-CS1 PE2-BC-CS2

Nucleic acids extraction from the filters using the Qiagen DNeasy Blood and Tissue Kit with some modifications to the manufacturer’s protocol. These extraction protocols are used by the University of South Florida (USF) for Florida Keys samples (FK) and the Monterey Bay Aquarium Research Institute (MBARI) for Monterey Bay, CA samples (MB). FK samples were extracted in the Breitbart Laboratory at the University of South Florida and all MB samples were extracted in the Chavez Laboratory at the Monterey Bay Aquarium Research Institute using the same extraction protocol.

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed. ispartof: Archives of Virology vol:160 issue:7 pages:1837-50 ispartof: location:Austria status: published

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Metabarcoding of environmental DNA (eDNA) samples holds new promise to increase our ability to measure changes in biodiversity and community composition over time. It can allow the characterization of large groups of organisms where traditional sampling may be impractical or not cost-effective. However, it is still unclear how best to compare and combine this information with morphological counts in order to inform policies and biodiversity metrics that are based on traditional sampling results. Under the Marine Biodiversity Observation Network (MBON) initiative, multiple taxonomic marker genes (16S rRNA, 18S rRNA, mitochondrial cytochrome c oxidase subunit I (COI), and 12S rRNA) have been used concurrently to examine the phylogenetic diversity of samples across trophic levels from microbes to vertebrates. Marker genes and their amplification primers target a different (and sometimes overlapping) group of organisms. Just as with traditional sampling methods, each have biases towards detecting certain organisms over others. Though eDNA metabarcoding often detects many more species than can be identified through microscopic or macroscopic net tow counts, processing and relating sequence data to traditional counts and biodiversity measures is an ongoing challenge. For samples collected within the MBON project, an analysis pipeline has been adapted to standardize sequence analysis of each marker gene. The pipeline processes reads from quality control and trimming through clustering of sequences into Operational Taxonomic Units (OTUs). Taxonomic identification of OTUs uses publically available sequence databases. Finally, the results of the analysis pipeline are combined into a Biological Observation Matrix (BIOM) file with metadata pertaining to the biological sample, PCR processing, and bioinformatic analysis. BIOM files can be used in downstream analysis to analyze biodiversity patterns within the samples. Monterey Bay in California, USA, is a hot spot of biodiversity and productivity fed by nutrient-rich upwelling water along the coast. A local time-series of samples has been collected by the Monterey Bay Aquarium Research Institute at coastal stations within the bay, providing several decades of contextual environmental data. Samples taken from this time series are ideal for testing the ability of eDNA sequencing to show variability in taxonomic groups over time. For metabarcoding analysis, samples were chosen representing different seasons corresponding to spring (early) upwelling, summer (late) upwelling, fall oceanic regime, and a winter (Davidson) regime from the years 2013-2016. Samples were analyzed across four taxonomic marker genes: two small-subunit ribosomal RNA genes targeting prokaryotic (16S rRNA) and eukaryotic (18S rRNA) organisms and two mitochondrial genes targeting eukaryotes (cytochrome c oxidase subunit I gene (COI)) and vertebrates (mitochondrial small-subunit ribosomal RNA gene (12S)). In order to combine data from multiple markers, species occupancy modeling was used to determine the probability that an OTU is truly present in a sample (as described in Kelly et al. 2017 and Lahoz-Monfort et al. 2015). Many taxonomic groups show seasonal trends in species abundance and diversity in Monterey Bay. Together this work illustrates the rewards and challenges of applying multiple genetic markers to eDNA sequencing analysis of an environmental time series.

The public has recently gained an increased appreciation of “good” or “friendly” bacteria, including commensal bacteria that are part of the healthy human microbiome (1) and bacteria present in common probiotic food products such as yogurt (2). Despite this new perception of bacteria, the term “virus” still has a predominantly negative connotation that is almost exclusively associated with disease (3). Consequently, the public frequently asks questions such as “Are there any good viruses?” In fact, there are many viruses that have mutualistic relationships with their hosts (4), but one viral group of particular interest for benefiting human health is the bacteriophages (phages: viruses that infect bacteria), which can specifically kill disease-causing bacterial pathogens. Battles between superheroes and villains easily capture the attention and enthusiasm of young learners, presenting an excellent opportunity to introduce interactions within the microbial world. This activity, which is appropriate for elementary-school children in a classroom or outreach setting, introduces learners to the concept of phage therapy, where certain phages (heroes) are used to destroy bacterial pathogens (villains) in a targeted and specific approach that does not negatively affect commensal bacteria (5). The specificity of phage therapy provides an advantage over antibiotic treatments, which disrupt the healthy microbiome. In addition, phage therapy presents an alternative strategy to treat bacteria that have developed resistance to antibiotics (6). In this activity, participants will learn about phage infection and host specificity by viewing photos of plaque assays performed in a laboratory with chromogenic bacteria, followed by a hands-on “fishing” activity using the phage heroes’ superpowers of specificity (hooks, Velcro, magnets) to capture particular bacterial villains. Templates are also provided for making custom temporary tattoos as activity rewards and for printing phage comic strips for a coloring activity.

The viruses circulating among Antarctic wildlife remain largely unknown. In an effort to identify viruses associated with Weddell seals (Leptonychotes weddellii) inhabiting the Ross Sea, vaginal and nasal swabs, and faecal samples were collected between November 2014 and February 2015. In addition, a Weddell seal kidney and South Polar skua (Stercorarius maccormicki) faeces were opportunistically sampled. Using high throughput sequencing, we identified and recovered 152 anellovirus genomes that share 63–70% genome-wide identities with other pinniped anelloviruses. Genome-wide pairwise comparisons coupled with phylogenetic analysis revealed two novel anellovirus species, tentatively named torque teno Leptonychotes weddellii virus (TTLwV) -1 and -2. TTLwV-1 (n = 133, genomes encompassing 40 genotypes) is highly recombinant, whereas TTLwV-2 (n = 19, genomes encompassing three genotypes) is relatively less recombinant. This study documents ubiquitous TTLwVs among Weddell seals in Antarctica with frequent co-infection by multiple genotypes, however, the role these anelloviruses play in seal health remains unknown.

Temperate bacterial viruses (phages) may enter a symbiosis with their host cell, forming a unit called a lysogen. Infection and viral replication are disassociated in lysogens until an induction event such as DNA damage occurs, triggering viral-mediated lysis. The lysogen-lytic viral reproduction switch is central to viral ecology, with diverse ecosystem impacts. It has been argued that lysogeny is favoured in phages at low host densities. This paradigm is based on the fraction of chemically inducible cells (FCIC) lysogeny proxy determined using DNA-damaging mitomycin C inductions. Contrary to the established paradigm, a survey of 39 inductions publications found FCIC to be highly variable and pervasively insensitive to bacterial host density at global, within-environment and within-study levels. Attempts to determine the source(s) of variability highlighted the inherent complications in using the FCIC proxy in mixed communities, including dissociation between rates of lysogeny and FCIC values. Ultimately, FCIC studies do not provide robust measures of lysogeny or consistent evidence of either positive or negative host density dependence to the lytic-lysogenic switch. Other metrics are therefore needed to understand the drivers of the lytic-lysogenic decision in viral communities and to test models of the host density-dependent viral lytic-lysogenic switch.

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Outnumbering all other biological entities on earth, bacteriophages (phages) play critical roles in structuring microbial communities through bacterial infection and subsequent lysis, as well as through horizontal gene transfer. While numerous studies have examined the effects of phages on free-living bacterial cells, much less is known regarding the role of phage infection in host-associated biofilms, which help to stabilize adherent microbial communities. Here we report the cultivation and characterization of a novel strain of Shewanella fidelis from the gut of the marine tunicate Ciona intestinalis, inducible prophages from the S. fidelis genome, and a strain-specific lytic phage recovered from surrounding seawater. In vitro biofilm assays demonstrated that lytic phage infection affects biofilm formation in a process likely influenced by the accumulation and integration of the extracellular DNA released during cell lysis, similar to the mechanism that has been previously shown for prophage induction.

Euphorbia mosaic virus (EuMV) was found in a symptomatic passionfruit ( Passiflora edulis ) plant from Homestead, Florida, USA, as well as in the symptomatic weed Euphorbia heterophylla . This is the first identification of EuMV in Florida and the United States and the first report of a natural infection of passionfruit by EuMV.

In light of water quality monitoring paradigms shifting to a more holistic approach, it is essential that environmental microbiologists embrace new methodological developments in clinical virology to create rapid, laboratory-free methods for the identification of wastewater pollution. It is widely accepted that routine monitoring of fecal indicator bacteria (FIB) does not adequately reflect human health risks associated with fecal pollution, especially risks posed by viruses. Enteric viruses are typically more resistant to wastewater treatment and persist longer in the environment than FIB. Furthermore, enteric viruses often have extremely low infectious doses. Currently, the incorporation of sanitary surveys, short-term monitoring of reference pathogens, exploratory quantitative microbial risk assessments, and predictive ecological models is being championed as the preferred approach to water management. In addition to improved virus concentration methods, simple, point-of-use tests for enteric viruses and/or improved viral indicators are needed to complement this emerging paradigm and ensure microbial safety worldwide.

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.

A genetic identification method (DNA barcoding) was used to compare the commu- nity compositions of planktonic fish eggs and larvae within a coastal embayment, testing the hypothesis that the composition of the planktonic larval fish community proportionately reflects the composition of the planktonic fish egg community (excluding species with non-planktonic eggs). By genetically identifying 843 individual eggs, we preserved the quantitative aspects of tra- ditional community analysis. The studied embayment has restricted hydrodynamic connectivity to other coastal waters. A circulation model containing simulated particles estimated average egg movement of approximately 1 km between times of spawning and sample collection, indicating locally spawned eggs were likely to be retained within the survey area. Thirteen of 14 collected egg taxa (88% of egg specimens) could be genetically identified to species level, with the 14th taxon identified to genus level. This novel approach revealed a high degree of spatial heterogene- ity in fish egg compositions within the embayment. Species that dominated the egg community (Eugerres plumieri, Cynoscion nebulosus, Centropomus undecimalis, and Prionotus spp.) were not particularly abundant amongst the 276 larvae identified, and the most abundant larval species (Achirus lineatus and Cynoscion arenarius) only comprised a minor proportion of the identified eggs. Overall, there was no correlation between the percent compositions of the egg and larval communities (r = −0.07, n = 15, p = 0.81). The clear disparities observed between the species com- positions of the egg and larvae highlight the need for directly identifying eggs when studying habitat connectivity or performing stock assessment with egg production model-based methods.

The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles.

We used a metagenomic approach to identify viruses that may be involved in the ecology of Daphnia spp. in Oneida and Cayuga lakes (upstate New York). We identified several highly represented, putative eukaryotic, circular, single-stranded deoxyribonucleic acid (DNA) viral genomes. Among these, we discovered a genotype similar in both sequence and genomic architecture to a virus previously reported from a hyperthermal lake that shares characteristics of both single-stranded ribonucleic acid (RNA) and single-stranded DNA viruses. We used quantitative polymerase chain reactions to study the prevalence and viral load of both positive-sense and negative-sense strands of the Daphnia mendotae–associated (Cladocera) hybrid virus (DMClaHV) over a summer season in Oneida and Cayuga lakes. DMClaHV had high prevalence within Daphnia populations, where viral load and the proportion of virus-positive individuals were higher preceding host population decline. DMClaHV viral load was different between two species of Daphnia (D. mendotae and D. retrocurva), and the dynamics between viruses and their hosts varied between the two lakes. We detected DMClaHV in eggs (ephippia) retrieved from Oneida Lake sediments with an estimated age of 30 yr. Using transmission electron microscopy, we observed small (20 nm diameter) virus-like particles in Daphnia that were well away from gut tissues and not associated with intracellular parasites. Because Daphnia plays a critical role in many lake ecosystems, DMClaHV may have important effects on herbivory and thus carbon flow through the lake ecosystem. More than two decades of investigation have highlighted the crucial importance of viruses to aquatic biogeochemistry and ecosystem function. Virus-like particles are highly abundant in plankton, and viruses of microorganisms cause considerable lysis, influencing elemental cycling, community diversity, and gene exchange among taxa (Fuhrman 1999; Weinbauer 2004; Suttle 2007). Despite the incorpo

To understand the similarities and differences between a free living viral population and its co-occurring temperate population, metagenomes of each type were prepared from the same seawater sample from Tampa Bay, FL. Libraries were prepared from extracted DNA of the ambient viruses and induced prophages from the co-occurring, viral-reduced microbial assemblage. Duplicate libraries were also prepared using the same DNA amplified by multiple displacement amplification. A non-viral-reduced, induced, amplified viral dataset from the same site in 2005 was reanalysed for temporal comparison. The induced viral metagenome was higher in identifiable virus sequences and differed from the other three datasets based on principal component, rarefaction, trinucleotide composition and contig spectrum analyses. This study indicated that induced prophages are unique and have lower overall community diversity than ambient viral populations from the same site. Both of the amplified contemporary metagenomes were enriched in single-stranded DNA (ssDNA) viral sequences. Six and 16 complete, circular ssDNA viral genomes were assembled from the amplified induced and ambient libraries, respectively, mostly similar to circoviruses. The amplified ambient metagenome contained genomes similar to an RNA-DNA hybrid virus recently identified in a hot spring and to an ssDNA virus infecting the diatom Chaetoceros.

A novel cyclovirus (proposed genus "Cyclovirus", family Circoviridae) was discovered in a specimen of Eurycotis floridana (Walker), also known as the Florida woods cockroach or palmetto bug, collected from Tarpon Springs, Florida. The Florida woods cockroach-associated cyclovirus GS140 (FWCasCyV-GS140) was obtained through a degenerate PCR assay and showed 64 % genome-wide pairwise identity to a cyclovirus identified in bat feces. This finding supports recent reports suggesting that Circoviridae members, traditionally thought to only infect vertebrates, are present within insect populations.

International audience; The family Circoviridae contains viruses with covalently closed, circular, single-stranded DNA (ssDNA) genomes, including the smallest known autonomously replicating, capsid-encoding animal pathogens. Members of this family are known to cause fatal diseases in birds and pigs and have been historically classified in one of two genera: Circovirus, which contains avian and porcine pathogens, and Gyrovirus, which includes a single species (Chicken anemia virus). However, over the course of the past six years, viral metagenomic approaches as well as degenerate PCR detection in unconventional hosts and environmental samples have elucidated a broader host range, including fish, a diversity of mammals, and invertebrates, for members of the family Circoviridae. Notably, these methods have uncovered a distinct group of viruses that are closely related to members of the genus Circovirus and comprise a new genus, Cyclovirus. The discovery of new viruses and a re-evaluation of genomic features that characterize members of the Circoviridae prompted a revision of the classification criteria used for this family of animal viruses. Here we provide details on an updated Circoviridae taxonomy ratified by the International Committee on the Taxonomy of Viruses in 2016, which establishes the genus Cyclovirus and reassigns the genus Gyrovirus to the family Anelloviridae, a separate lineage of animal viruses that also contains circular ssDNA genomes. In addition, we provide a new species demarcation threshold of 80% genome-wide pairwise identity for members of the family Circoviridae, based on pairwise identity distribution analysis, and list guidelines to distinguish between members of this family and other eukaryotic viruses with circular, ssDNA genomes.

Current microbial water quality monitoring is generally limited to culture-based measurements of fecal indicator bacteria (FIB). Given the many possible sources of fecal pollution within a watershed and extra-intestinal FIB reservoirs, it is important to determine source(s) of fecal pollution as a means to improve water quality and protect public health. The principal objective of this investigation was to characterize the microbial water quality of shellfish harvesting areas in the Gulf of Nicoya, Costa Rica during 2015. In order to achieve this objective, the specificity and sensitivity of 11 existing microbial source tracking (MST) PCR assays, associated with cows (BacCow), dogs (BacCan, DogBac), domestic wastewater (PMMoV), general avian (GFD), gulls (Gull2), horses (HorseBac, HoF), humans (HF183, HPyV), and pigs (PF), were evaluated using domestic wastewater and animal fecal samples collected from the region. The sensitivity of animal-associated assays ranged from 13 to 100%, while assay specificity ranged from 38 to 100%. The specificity of pepper mild mottle virus (PMMoV) and human polyomavirus (HPyV) was 100% for domestic wastewater, as compared to 94% specificity of the HF183 Bacteroidales marker. PMMoV was identified as a useful domestic wastewater-associated marker, with concentrations as high as 1.1 × 105 copies/ml and 100% sensitivity and specificity. Monthly surface water samples collected from four shellfish harvesting areas were analyzed using culture-based methods for Escherichia coli as well as molecular methods for FIB and a suite of MST markers, which were selected for their specificity in the region. While culturable E. coli results suggested possible fecal pollution during the monitoring period, the absence of human/domestic wastewater-associated markers and low FIB concentrations determined using molecular methods indicated sufficient microbial water quality for shellfish harvesting. This is the first study to our knowledge to test the performance of MST markers in Costa Rica as well as in Central America. Given the lack of wastewater treatment and the presence of secondary sources of FIB, this study highlights the importance of an MST toolbox approach to characterize water quality in tropical regions. Furthermore, it confirms and extends the geographic range of PMMoV as an effective tool for monitoring domestic wastewater pollution.

Aims To identify faecal pollution along the southeastern Florida coast and determine the performance of a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) method for pepper mild mottle virus (PMMoV). Methods and Results In 2014, bimonthly surface water samples were collected from inlets, exposed to runoff and septic seepage, and coastal sites, exposed to ocean outfalls. Analysis of culturable enterococci and a suite of microbial source tracking (MST) markers (BacHum, CowM2, DogBact, HF183, HPyV, PMMoV) revealed faecal pollution, primarily of human origin, at all sites. Since PMMoV was detected more frequently than other MST markers, the process limits of quantification (undiluted to 10-2 dilution) and detection (10-2 dilution) for the RT-qPCR method were determined by seeding untreated wastewater into the coastal waters. Simulated quantitative microbial risk assessment, employing human norovirus as a reference pathogen, calculated a 0.286 median risk of gastrointestinal illness associated with the PMMoV limit of detection. Conclusions All sites met the U.S. EPA recreational water criteria, despite detection of domestic astewater associated MST markers. PMMoV correlated only with human-associated MST markers. Significance and Impact of Study This study demonstrated that PMMoV is an important domestic wastewater-associated marker that should be included in the MST toolbox; therefore, future studies should thoroughly investigate the health risks associated with its detection and quantification in environmental waters. This article is protected by copyright. All rights reserved.

Conventional wastewater treatment plant (WWTP) processes are primarily designed to reduce the amount of organic matter, pathogens, and nutrients from the incoming influent. However, these processes are not as effective in reducing the concentrations of micropollutants, including endocrine-disrupting compounds (EDCs), which notoriously evade traditional wastewater treatment technologies and are found even in tertiary-treated effluent. For WWTPs practicing deep-well injection or surface-water discharge, EDCs in the treated effluent are discharged into groundwater or the aquatic environment where humans and wildlife may potentially suffer the effects of chemical exposure. In the current laboratory-scale study, we tested a bench-top electrocoagulation (EC) unit utilizing aluminum blades for the removal of six estrogenic EDCs [estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), bisphenol-A (BPA), and nonylphenol (NP)]. Samples of municipal wastewater influent and tertiary-treated effluent were spiked with the six EDCs in order to test the removal efficiency of the EC unit. The mean concentration of each EDC component was statistically lower after EC treatment (removal range = 42%–98%). To our knowledge, this is the first study to investigate aluminum electrocoagulation for removal of these specific EDCs, including nonylphenol (without the ethoxylate chain), as well as natural and synthetic estrogens.

Limited supply of clean water in urbanizing watersheds creates challenges for safely sustaining irrigated agriculture and global food security. On-farm interventions, such as riverbank filtration (RBF), are used in developing countries to treat irrigation water from rivers with extensive fecal contamination. Using a Bayesian approach incorporating ethnographic data and pathogen measurements, quantitative microbial risk assessment (QMRA) methods were employed to assess the impact of RBF on consumer health burdens for Giardia, Cryptosporidium, rotavirus, norovirus, and adenovirus infections resulting from indirect wastewater reuse, with lettuce irrigation in Bolivia as a model system. Concentrations of the microbial source tracking markers pepper mild mottle virus and HF183 Bacteroides were respectively 2.9 and 5.5 log10 units lower in RBF-treated water than in the river water. Consumption of lettuce irrigated with river water caused an estimated median health burden that represents 37% of Bolivia's overall diarrheal disease burden, but RBF resulted in an estimated health burden that is only 1.1% of this overall diarrheal disease burden. Variability and uncertainty associated with environmental and cultural factors affecting exposure correlated more with QMRA-predicted health outcomes than factors related to disease vulnerability. Policies governing simple on-farm interventions like RBF can be intermediary solutions for communities in urbanizing watersheds that currently lack wastewater treatment.

Monopartite begomoviruses (Geminiviridae), which are whitefly-transmitted single-stranded DNA viruses known for causing devastating crop diseases, are often associated with satellite DNAs. Since begomovirus acquisition or exchange of satellite DNAs may lead to adaptation to new plant hosts and emergence of new disease complexes, it is important to investigate the diversity and distribution of these molecules. This study reports begomovirus-associated satellite DNAs identified during a vector-enabled metagenomic (VEM) survey of begomoviruses using whiteflies collected in various locations (California (USA), Guatemala, Israel, Puerto Rico, and Spain). Protein-encoding satellite DNAs, including alphasatellites and betasatellites, were identified in Israel, Puerto Rico, and Guatemala. Novel alphasatellites were detected in samples from Guatemala and Puerto Rico, resulting in the description of a phylogenetic clade (DNA-3-type alphasatellites) dominated by New World sequences. In addition, a diversity of small (~640–750 nucleotides) satellite DNAs similar to satellites associated with begomoviruses infecting Ipomoea spp. were detected in Puerto Rico and Spain. A third class of satellite molecules, named gammasatellites, is proposed to encompass the increasing number of reported small (, This project is funded by grants from the National Science Foundation’s Systematic Biology and Biodiversity Inventories Program (DEB-1025915 to MB, DEB-1025901 to JEP). Authors would like to thank all the collaborators who provided whitefly specimens, including Jorge Gonzales (Guatemala), Moshe Lapidot (Israel), Bill Wintermantel (USA), James D. McCright (USA), Eric Portales (Guatemala), Esvin Salguero (Guatemala), Sakata Seed of Guatemala, and Sakata Seed of South America, We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

BackgroundViruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).MethodsHere we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.ResultsMock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (Microviridaefamily, can be among the most abundant viral genomes in a sample.DiscussionTogether these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Residing in a phylum of their own, ctenophores are gelatinous zooplankton that drift through the ocean's water column. Although ctenophores are known to be parasitized by a variety of eukaryotes, no studies have examined their bacterial associates. This study describes the bacterial communities associated with the lobate ctenophore Mnemiopsis leidyi and its natural predator Beroe ovata in Tampa Bay, Florida, USA. Investigations using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes demonstrated that ctenophore bacterial communities were distinct from the surrounding water. In addition, each ctenophore genus contained a unique microbiota. Ctenophore samples contained fewer bacterial operational taxonomic units (OTUs) by T-RFLP and lower diversity communities by 16S rRNA gene sequencing than the water column. Both ctenophore genera contained sequences related to bacteria previously described in marine invertebrates, and sequences similar to a sea anemone pathogen were abundant in B. ovata. Temporal sampling revealed that the ctenophore-associated bacterial communities varied over time, with no single OTU detected at all time points. This is the first report of distinct and dynamic bacterial communities associated with ctenophores, suggesting that these microbial consortia may play important roles in ctenophore ecology. Future work needs to elucidate the functional roles and mode of acquisition of these bacteria.