Macroalgal virosphere assists with host-microbiome equilibrium regulation and affects prokaryotes in surrounding marine environments.

The microbiomes in macroalgal holobionts play vital roles in regulating macroalgal growth and ocean carbon cycling. However, the virospheres in macroalgal holobionts remain largely underexplored, representing a critical knowledge gap. Here we unveil that the holobiont of kelp (Saccharina japonica) harbors highly specific and unique epiphytic/endophytic viral species, with novelty (99.7% unknown) surpassing even extreme marine habitats (e.g. deep-sea and hadal zones), indicating that macroalgal virospheres, despite being closest to us, are among the least understood. These viruses potentially maintain microbiome equilibrium critical for kelp health via lytic-lysogenic infections and the expression of folate biosynthesis genes. In-situ kelp mesocosm cultivation and metagenomic mining revealed that kelp holobiont profoundly reshaped surrounding seawater and sediment virus-prokaryote pairings through changing surrounding environmental conditions and virus-host migrations. Some kelp epiphytic viruses could even infect sediment autochthonous bacteria after deposition. Moreover, the presence of ample viral auxiliary metabolic genes for kelp polysaccharide (e.g. laminarin) degradation underscores the underappreciated viral metabolic influence on macroalgal carbon cycling. This study provides key insights into understanding the previously overlooked ecological significance of viruses within macroalgal holobionts and the macroalgae-prokaryotes-virus tripartite relationship.

Isolation and Characterization of the First Temperate Virus Infecting Psychrobacillus from Marine Sediments.

Viruses are far more abundant than cellular microorganisms in the marine ecosystem. However, very few viruses have so far been isolated from marine sediments, especially hydrothermal vent sediments, hindering the understanding of the biology and ecological functions of these tiny organisms. Here, we report the isolation and characterization of a temperate bacteriophage, named PVJ1, which infects Psychrobacillus from a hydrothermal vent field in Okinawa Trough. PVJ1 belongs to the Myoviridae family of the order Caudovirales. The tailed phage possesses a 53,187 bp linear dsDNA genome, with 84 ORFs encoding structural proteins, genome replication, host lysis, etc. in a modular pattern. The phage genome is integrated into the host chromosome near the 3'-end of deoD, a gene encoding purine nucleoside phosphorylase (PNP). The phage integration does not appear to disrupt the function of PNP. The phage DNA is packaged by the headful mechanism. Release of PVJ1 from the host cell was drastically enhanced by treatment with mitomycin C. Phages encoding an MCP sharing significant similarity (≥70% identical amino acids) with that of PVJ1 are widespread in diverse environments, including marine and freshwater sediments, soils, artificial ecosystems, and animal intestines, and primarily infect Firmicutes. These results are valuable to the understanding of the lifestyle and host interactions of bacterial viruses at the bottom of the ocean.

Persistence of poliovirus types 2 and 3 in waste-impacted water and sediment.

Eradication of poliovirus (PV) is a global public health priority, and as clinical cases decrease, the role of environmental surveillance becomes more important. Persistence of PV and the environmental factors that influence it (such as temperature and sample type) are an important part of understanding and interpreting positive environmental surveillance samples. The objective of this study was to evaluate the persistence of poliovirus type 2 (PV2) and type 3 (PV3) in wastewater and sediment. Microcosms containing either 1) influent wastewater or 2) influent wastewater with a sediment matrix were seeded with either PV2 or PV3, and stored for up to 126 days at three temperatures (4°C, room temperature [RT], and 30°C). Active PV in the liquid of (1), and the sediment and liquid portions of (2) were sampled and quantified at up to 10 time points via plaque assay and RT-qPCR. A suite of 17 models were tested for best fit to characterize decay of PV2 and PV3 over time and determine the time points at which >90% (T90) and >99% (T99) reduction was reached. Linear models assessed the influence of experimental factors (matrix, temperature, virus type and method of detection) on the predicted T90 and T99 values. Results showed that when T90 was the dependent variable, virus type, matrix, and temperature significantly affected decay, and there was a clear interaction between the sediment matrix and temperature. When T99 was the dependent variable, only temperature and matrix type significantly influenced the decay metric. This study characterizes the persistence of both active and molecular PV2 and PV3 in relevant environmental conditions, and demonstrates that temperature and sediment both play important roles in PV viability. As eradication nears and clinical cases decrease, environmental surveillance and knowledge of PV persistence will play a key role in understanding the silent circulation in endemic countries.

Revealing the Viral Community in the Hadal Sediment of the New Britain Trench.

Marine viruses are widely distributed and influence matter and energy transformation in ecosystems by modulating hosts' metabolism. The hadal trenches represent the deepest marine habitat on Earth, for which the viral communities and related biogeochemical functions are least explored and poorly understood. Here, using the sediment samples (8720 m below sea level) collected from the New Britain Trench (NBT), we investigated the viral community, diversity, and genetic potentials in the hadal sediment habitat for the first time by deep shotgun metagenomic sequencing. We found the NBT sediment viral community was dominated by Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, and Phycodnaviridae, which belong to the dsDNA viruses. However, the large majority of them remained uncharacterized. We found the hadal sediment virome had some common components by comparing the hadal sediment viruses with those of hadal aquatic habitats and those of bathypelagic and terrestrial habitats. It was also distinctive in community structure and had many novel viral clusters not associated with the other habitual virome included in our analyses. Further phylogenetic analysis on its Caudovirales showed novel diversities, including new clades specially evolved in the hadal sediment habitat. Annotation of the NBT sediment viruses indicated the viruses might influence microbial hydrocarbon biodegradation and carbon and sulfur cycling via metabolic augmentation through auxiliary metabolic genes (AMGs). Our study filled in the knowledge gaps on the virome of the hadal sediment habitats and provided insight into the evolution and the potential metabolic functions of the hadal sediment virome.

Revealing the full biosphere structure and versatile metabolic functions in the deepest ocean sediment of the Challenger Deep.

BACKGROUND: The full biosphere structure and functional exploration of the microbial communities of the Challenger Deep of the Mariana Trench, the deepest known hadal zone on Earth, lag far behind that of other marine realms. RESULTS: We adopt a deep metagenomics approach to investigate the microbiome in the sediment of Challenger Deep, Mariana Trench. We construct 178 metagenome-assembled genomes (MAGs) representing 26 phyla, 16 of which are reported from hadal sediment for the first time. Based on the MAGs, we find the microbial community functions are marked by enrichment and prevalence of mixotrophy and facultative anaerobic metabolism. The microeukaryotic community is found to be dominated by six fungal groups that are characterized for the first time in hadal sediment to possess the assimilatory and dissimilatory nitrate/sulfate reduction, and hydrogen sulfide oxidation pathways. By metaviromic analysis, we reveal novel hadal Caudovirales clades, distinctive virus-host interactions, and specialized auxiliary metabolic genes for modulating hosts' nitrogen/sulfur metabolism. The hadal microbiome is further investigated by large-scale cultivation that cataloged 1070 bacterial and 19 fungal isolates from the Challenger Deep sediment, many of which are found to be new species specialized in the hadal habitat. CONCLUSION: Our hadal MAGs and isolates increase the diversity of the Challenger Deep sediment microbial genomes and isolates present in the public. The deep metagenomics approach fills the knowledge gaps in structure and diversity of the hadal microbiome, and provides novel insight into the ecology and metabolism of eukaryotic and viral components in the deepest biosphere on earth.

Cyanophage Diversity and Community Structure in Dead Zone Sediments.

Up to 20% of prokaryotic organisms in the oceans are estimated to die every day due to viral infection and lysis. Viruses can therefore alter microbial diversity, community structure, and biogeochemical processes driven by these organisms. Cyanophages are viruses that infect and lyse cyanobacterial cells, adding bioavailable carbon and nutrients into the environment. Cyanobacteria are photosynthesizing bacteria, with some species capable of N2 fixation, which are known to form large blooms as well as resistant resting cells known as akinetes. Here, we investigated cyanophage diversity and community structure plus cyanobacteria in dead zone sediments. We sampled surface sediments and sequenced DNA and RNA, along an oxygen gradient-representing oxic, hypoxic, and anoxic conditions-in one of the world's largest dead zones located in the Baltic Sea. Cyanophages were detected at all stations and, based on partial genome contigs, had a higher alpha diversity and different beta diversity in the hypoxic-anoxic sediments, suggesting that cyanobacteria in dead zone sediments and/or environmental conditions select for specific cyanophages. Some of these cyanophages can infect cyanobacteria with potential consequences for gene expression related to their photosystem and phosphate regulation. Top cyanobacterial genera detected in the anoxic sediment included Dolichospermum/Anabaena, Synechococcus, and Cyanobium RNA transcripts classified to cyanobacteria were associated with numerous pathways, including anaerobic carbon metabolism and N2 fixation. Cyanobacterial blooms are known to fuel oxygen-depleted ecosystems with phosphorus (so-called internal loading), and our cyanophage data indicate the potential for viral lysis of cyanobacteria which might explain the high nutrient turnover in these environments.IMPORTANCE Cyanophages are viruses that target cyanobacteria and directly control their abundance via viral lysis. Cyanobacteria are known to cause large blooms in water bodies, substantially contributing to oxygen depletion in bottom waters resulting in areas called dead zones. Our knowledge of cyanophages in dead zones is very scarce, and so far, no studies have assembled partial cyanophage genomes and investigated their associated cyanobacteria in these dark and anoxic sediments. Here, we present the first study using DNA and RNA sequencing to investigate in situ diversity of cyanophages and cyanobacteria in dead zones. Our study shows that dead zone sediments contain different cyanophages compared to oxic sediments and suggest that these viruses are able to affect cyanobacterial photosystem and phosphate regulation. Furthermore, cyanophage-controlled lysis of cyanobacteria might also increase the turnover of carbon, phosphorus, and nitrogen in these oxygen-free environments at the bottom of the sea.

An Advanced Protocol for the Quantification of Marine Sediment Viruses via Flow Cytometry.

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R2 = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Sediment re-suspension as a potential mechanism for viral and bacterial contaminants.

Pathogenic enteric viruses and bacteria tend to occur in higher concentrations and survive longer in aquatic sediments than suspended in the water column. Re-suspension of these organisms can result in a significant degradation of overlying water quality. Additionally, the re-suspension of microbial pathogens in artificial irrigation canals could endanger the consumption of fresh and ready-to-eat produce. Irrigation water has been implicated in numerous fresh produce outbreaks over the last 30 years. This study aimed to quantify the proportions of bacterial and viral re-suspension from sediment in a recirculating flume with varying velocities. MS2 coliphage and Escherichia coli were found to re-suspend at rates that were not significantly different, despite organism size differences. However, E. coli re-suspension rates from sand and clay were significantly different. This suggests that likely sediment-associated particles were recovered with the organisms attached. Similar re-suspension rates are hypothesized to be due to the dynamics of sediment transport, rather than that of the organisms themselves. This study also indicated that the re-suspension of sediment at very low velocities (e.g., less than 10 cm/s), could impact the microbiological quality of the overlaying water. Results from this study conclude that sediment could be a viable mechanism for irrigation water contamination.

Quantitative Detection of Human Adenovirus and Human Rotavirus Group A in Wastewater and El-Rahawy Drainage Canal Influencing River Nile in the North of Giza, Egypt.

Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this study, the prevalence of human adenovirus (HAdV), rotavirus group A (RVA) was determined in urban sewage, activated sludge, drainage water, drainage sediment, Nile water, and Nile sediment, using quantitative polymerase chain reaction (qPCR) analysis. HAdV was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 genome copies/liter (GC/L), 33% of activated sludge with viral concentrations ranging from 103 to 107 GC/kilogram (GC/kg), 95% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 75% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, 50% of Nile water with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile sediment with viral concentrations ranging from 103 to 107 GC/kg. RVA was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 GC/L, 75% of activated sludge with viral concentrations ranging from 103 to 107 GC/L, 58% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 50% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile water with viral concentrations ranging from 103 to 107 GC/kg. In conclusion, Abu-Rawash WWTP acts as a source of HAdV and RVA, releasing them into El-Rahawy drain then to the River Nile Rosetta branch.

Dead or alive: sediment DNA archives as tools for tracking aquatic evolution and adaptation.

DNA can be preserved in marine and freshwater sediments both in bulk sediment and in intact, viable resting stages. Here, we assess the potential for combined use of ancient, environmental, DNA and timeseries of resurrected long-term dormant organisms, to reconstruct trophic interactions and evolutionary adaptation to changing environments. These new methods, coupled with independent evidence of biotic and abiotic forcing factors, can provide a holistic view of past ecosystems beyond that offered by standard palaeoecology, help us assess implications of ecological and molecular change for contemporary ecosystem functioning and services, and improve our ability to predict adaptation to environmental stress.

TARGETED RESEQUENCING OF WETLAND SEDIMENT AS A TOOL FOR AVIAN INFLUENZA VIRUS SURVEILLANCE.

Surveillance methods for avian influenza virus (AIV) based upon collecting and testing samples from individual wild birds have several significant limitations primarily related to the difficulties associated with obtaining samples. Because AIVs are shed in waterfowl feces, the use of environmental substrates where waterfowl feces accumulate may overcome some of these limitations. However, these substrates are difficult to analyze using traditional diagnostic techniques, such as virus culture and PCR, because of virus inactivation, RNA degradation, low concentration of target RNA, microbial complexity, presence of inhibitory substances, and other factors. We investigated the use of a genomics-based approach called targeted resequencing to detect and characterize AIVs in wetland sediments during the 2014-15 North American highly pathogenic avian influenza outbreak. We identified AIV in 20.6% (71/345) sediment samples obtained from wetlands (n=15) and outdoor waterbodies on AIV-infected poultry farms (n=10) in British Columbia, Canada (the first area affected during the outbreak). Thirteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes were detected, including H5, N1, and N8 sequences that clustered with other sequences associated with the North American outbreak. Additionally, as many as eight HA and eight NA subtypes could be detected in a single sediment sample. This proof-of-concept study shows the potential utility of sediment sampling coupled with genomics-based analysis as a tool for AIV surveillance.

Human mastadenovirus in water, sediment, sea surface microlayer, and bivalve mollusk from southern Brazilian beaches.

Anthropogenic contamination of beaches in the south of Brazil was assessed by detection of Escherichia coli, human mastadenovirus species C (HAdV-C) and F (HAdV-F) and hepatitis E virus (HEV). Sampling was carried out in October (2016), and in January, April and July (2017). Water, sediment, sea surface microlayer (SML), bivalves, and air sentinel samples were evaluated. Quantitative microbiological risk assessment (QMRA) was used to estimate the probability of swimmer infection. HAdV-C was present in 26% of the samples, for both qPCR and viral isolation. The highest rates of detection in genomic copies (GC) were in water (2.42E+10 GC/L), SML (2.08E+10 GC/L), sediment (3.82E+08 GC/g) and bivalves (3.91E+07 GC/g). QMRA estimated daily and annual risks with a maximum value (9.99E-01) in almost all of the samples. Viable HAdV-C was often detected in the SML, pointing that this is a source of infection for people bathing in these waters.

eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species' activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Molecular Diversity of Cyanopodoviruses in Two Coastal Wetlands in Northeast China.

Although bacteriophages are the most abundant biological entities on the planet, their genetic diversity, especially in natural wetlands, is poorly understood. In this study, the genetic diversity of cyanopodoviruses in sediments of two coastal wetlands in Northeast China was investigated by targeting the DNA polymerase (pol) gene. A total of 66 DNA pol clones were obtained. A BLAST search at the amino acid level showed that the obtained sequences had the highest identity ranged from 83 to 99% to the known sequences. A phylogenetic tree showed that the distribution patterns of DNA pol sequence were different between two wetland soils, and 29 clones of this study formed four wetland-specific groups, which suggested that unrevealed novel groups of cyanopodovirus inhabited in wetlands. In addition, nonmetric multidimensional scaling (NMDS) analysis of all DNA pol sequences from various environments showed that cyanopodovirus communities of coastal wetlands are in the intermediate position between marine water environments and terrestrial freshwater environments, which highlights that the coastal wetlands as transitional zones between inland freshwater environments and marine environments.

Recovery of influenza A viruses from lake water and sediments by experimental inoculation.

Influenza A viruses (IAV) are zoonotic pathogens relevant to human, domestic animal and wildlife health. Many avian IAVs are transmitted among waterfowl via a faecal-oral-route. Therefore, environmental water where waterfowl congregate may play an important role in the ecology and epidemiology of avian IAV. Water and sediment may sustain and transmit virus among individuals or species. It is unclear at what concentrations waterborne viruses are infectious or remain detectable. To address this, we performed lake water and sediment dilution experiments with varying concentrations or infectious doses of four IAV strains from seal, turkey, duck and gull. To test for infectivity of the IAV strains in a concentration dependent manner, we applied cultivation to specific pathogen free (SPF) embryonated chicken eggs and Madin-Darby Canine Kidney (MDCK) cells. IAV recovery was more effective from embryonated chicken eggs than MDCK cells for freshwater lake dilutions, whereas, MDCK cells were more effective for viral recovery from sediment samples. Low infectious dose (1 PFU/200 µL) was sufficient in most cases to detect and recover IAV from lake water dilutions. Sediment required higher initial infectious doses (≥ 100 PFU/200 µL).

Virus Genomes from Deep Sea Sediments Expand the Ocean Megavirome and Support Independent Origins of Viral Gigantism.

The nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes (proposed order, "Megavirales") include the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, and Mimiviridae, as well as still unclassified pithoviruses, pandoraviruses, molliviruses, and faustoviruses. Several of these virus groups include giant viruses, with genome and particle sizes exceeding those of many bacterial and archaeal cells. We explored the diversity of the NCLDV in deep sea sediments from the Loki's Castle hydrothermal vent area. Using metagenomics, we reconstructed 23 high-quality genomic bins of novel NCLDV, 15 of which are related to pithoviruses, 5 to marseilleviruses, 1 to iridoviruses, and 2 to klosneuviruses. Some of the identified pithovirus-like and marseillevirus-like genomes belong to deep branches in the phylogenetic tree of core NCLDV genes, substantially expanding the diversity and phylogenetic depth of the respective groups. The discovered viruses, including putative giant members of the family Marseilleviridae, have a broad range of apparent genome sizes, in agreement with the multiple, independent origins of gigantism in different branches of the NCLDV. Phylogenomic analysis reaffirms the monophyly of the pithovirus-iridovirus-marseillevirus branch of the NCLDV. Similarly to other giant viruses, the pithovirus-like viruses from Loki's Castle encode translation systems components. Phylogenetic analysis of these genes indicates a greater bacterial contribution than had been detected previously. Genome comparison suggests extensive gene exchange between members of the pithovirus-like viruses and Mimiviridae Further exploration of the genomic diversity of Megavirales in additional sediment samples is expected to yield new insights into the evolution of giant viruses and the composition of the ocean megavirome.IMPORTANCE Genomics and evolution of giant viruses are two of the most vigorously developing areas of virus research. Lately, metagenomics has become the main source of new virus genomes. Here we describe a metagenomic analysis of the genomes of large and giant viruses from deep sea sediments. The assembled new virus genomes substantially expand the known diversity of the nucleocytoplasmic large DNA viruses of eukaryotes. The results support the concept of independent evolution of giant viruses from smaller ancestors in different virus branches.

Sediments from Arctic Tide-Water Glaciers Remove Coastal Marine Viruses and Delay Host Infection.

Over the past few decades, the Arctic region has been strongly affected by global warming, leading to increased sea surface temperatures and melting of land and sea ice. Marine terminating (tide-water) glaciers are expected to show higher melting and calving rates, with an increase in the input of fine sediment particles in the coastal marine environment. We experimentally investigated whether marine viruses, which drive microbial interactions and biogeochemical cycling are removed from the water column through adsorption to glacier-delivered fine sediments. Ecologically relevant concentrations of 30, 100 and 200 mg·L-1 sediments were added to filtered lysates of 3 cultured algal viruses and to a natural marine bacterial virus community. Total virus removal increased with sediment concentration whereby the removal rate depended on the virus used (up to 88% for an Arctic algal virus), suggesting a different interaction strength with the sediment. Moreover, we observed that the adsorption of viruses to sediment is a reversible process, and that desorbed viruses are still able to infect their respective hosts. Nonetheless, the addition of sediment to infection experiments with the Arctic prasinovirus MpoV-45T substantially delayed host lysis and the production of progeny viruses. We demonstrate that glacier-derived fine sediments have the potency to alter virus availability and consequently, host population dynamics.

Critical Evaluation of CrAssphage as a Molecular Marker for Human-Derived Wastewater Contamination in the Aquatic Environment.

The discharge of human-derived wastewater represents a major threat to water quality with the potential for waterborne disease outbreaks mainly associated with enteric viruses. To prevent illnesses, indicators associated with fecal contamination are monitored in polluted areas, however, their prevalence often does not correlate well with viral pathogens. In this study, we used crAssphage, a recently discovered human-specific gut-associated bacteriophage, for the surveillance of wastewater-derived viral contamination. Untreated and treated wastewater, surface water, sediment and mussel samples were collected monthly over 1 year from the Conwy River and estuary (UK) and were analyzed for crAssphage marker by quantitative PCR. This is the first long-term catchment-to-coast scale study of environmental crAssphage concentrations. CrAssphage was detected in all sample types and showed no distinct seasonal pattern. CrAssphage concentrations were 2 × 105-109 genome copies (gc)/L in all untreated wastewater influent and 107-108 gc/L in secondary treated effluent samples, 3 × 103 gc/L-3 × 107 gc/L in surface water samples (94% positive) and 2 × 102-104 gc/g sediment (68% positive) and mussel digestive tissue (79% positive). CrAssphage concentrations were 1-5 log10 higher than human enteric virus titers (norovirus, sapovirus, adenovirus, polyomavirus). Our results indicate that crAssphage is well suited to tracking human wastewater contamination and pollution risk assessment in aquatic environments.

Influence of physico-chemical characteristics of sediment on the in situ spatial distribution of F-specific RNA phages in the riverbed.

Riverbed sediment is commonly described as an enteric virus reservoir and thought to play an important role in water column contamination, especially during rainfall events. Although the occurrence and fate of faecal-derived viruses are fairly well characterized in water, little information is available on their presence as their interactions with sediment. This study aimed at determining the main environmental factors responsible for the presence of enteric viruses in riverbed sediment. Using a combination of microbiological and physico-chemical analyses of freshly field-sampled sediments, we demonstrated their contamination by faecal phages. The in situ spatial distribution of phages in sediment was mainly driven by sediment composition. A preferential phage accumulation occurred along one bank of the river, where the quantity of fine sands and clay particles smaller than 0.2 mm was the highest. Additionally, a mineralogical analysis revealed the influence of the heterogeneous presence of virus sorbents such as quartz, calcite, carbonates and iron-bearing phases (goethite) on the phage spatial pattern. A more precise knowledge of the composition of riverbed sediment is therefore useful for predicting preferential areas of enteric virus accumulation and should allow more accurate microbial risk assessment when using surface water for drinking water production or recreational activities.