Limited potexvirus diversity in eastern Gulf of Mexico seagrass meadows.

Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity affects host-virus interactions.

Simultaneous detection of pathogens and antimicrobial resistance genes with the open source, cloud-based, CZ ID pipeline.

Antimicrobial resistant (AMR) pathogens represent urgent threats to human health, and their surveillance is of paramount importance. Metagenomic next generation sequencing (mNGS) has revolutionized such efforts, but remains challenging due to the lack of open-access bioinformatics tools capable of simultaneously analyzing both microbial and AMR gene sequences. To address this need, we developed the CZ ID AMR module, an open-access, cloud-based workflow designed to integrate detection of both microbes and AMR genes in mNGS and whole-genome sequencing (WGS) data. It leverages the Comprehensive Antibiotic Resistance Database and associated Resistance Gene Identifier software, and works synergistically with the CZ ID short-read mNGS module to enable broad detection of both microbes and AMR genes. We highlight diverse applications of the AMR module through analysis of both publicly available and newly generated mNGS and WGS data from four clinical cohort studies and an environmental surveillance project. Through genomic investigations of bacterial sepsis and pneumonia cases, hospital outbreaks, and wastewater surveillance data, we gain a deeper understanding of infectious agents and their resistomes, highlighting the value of integrating microbial identification and AMR profiling for both research and public health. We leverage additional functionalities of the CZ ID mNGS platform to couple resistome profiling with the assessment of phylogenetic relationships between nosocomial pathogens, and further demonstrate the potential to capture the longitudinal dynamics of pathogen and AMR genes in hospital acquired bacterial infections. In sum, the new AMR module advances the capabilities of the open-access CZ ID microbial bioinformatics platform by integrating pathogen detection and AMR profiling from mNGS and WGS data. Its development represents a critical step toward democratizing pathogen genomic analysis and supporting collaborative efforts to combat the growing threat of AMR.

Simultaneous detection of pathogens and antimicrobial resistance genes with the open source, cloud-based, CZ ID pipeline.

Antimicrobial resistant (AMR) pathogens represent urgent threats to human health, and their surveillance is of paramount importance. Metagenomic next generation sequencing (mNGS) has revolutionized such efforts, but remains challenging due to the lack of open-access bioinformatics tools capable of simultaneously analyzing both microbial and AMR gene sequences. To address this need, we developed the Chan Zuckerberg ID (CZ ID) AMR module, an open-access, cloud-based workflow designed to integrate detection of both microbes and AMR genes in mNGS and whole-genome sequencing (WGS) data. It leverages the Comprehensive Antibiotic Resistance Database and associated Resistance Gene Identifier software, and works synergistically with the CZ ID short-read mNGS module to enable broad detection of both microbes and AMR genes. We highlight diverse applications of the AMR module through analysis of both publicly available and newly generated mNGS and WGS data from four clinical cohort studies and an environmental surveillance project. Through genomic investigations of bacterial sepsis and pneumonia cases, hospital outbreaks, and wastewater surveillance data, we gain a deeper understanding of infectious agents and their resistomes, highlighting the value of integrating microbial identification and AMR profiling for both research and public health. We leverage additional functionalities of the CZ ID mNGS platform to couple resistome profiling with the assessment of phylogenetic relationships between nosocomial pathogens, and further demonstrate the potential to capture the longitudinal dynamics of pathogen and AMR genes in hospital acquired bacterial infections. In sum, the new AMR module advances the capabilities of the open-access CZ ID microbial bioinformatics platform by integrating pathogen detection and AMR profiling from mNGS and WGS data. Its development represents a critical step toward democratizing pathogen genomic analysis and supporting collaborative efforts to combat the growing threat of AMR.

Freshwater macrophytes harbor viruses representing all five major phyla of the RNA viral kingdom Orthornavirae.

Research on aquatic plant viruses is lagging behind that of their terrestrial counterparts. To address this knowledge gap, here we identified viruses associated with freshwater macrophytes, a taxonomically diverse group of aquatic phototrophs that are visible with the naked eye. We surveyed pooled macrophyte samples collected at four spring sites in Florida, USA through next generation sequencing of RNA extracted from purified viral particles. Sequencing efforts resulted in the detection of 156 freshwater macrophyte associated (FMA) viral contigs, 37 of which approximate complete genomes or segments. FMA viral contigs represent putative members from all five major phyla of the RNA viral kingdom Orthornavirae. Similar to viral types found in land plants, viral sequences identified in macrophytes were dominated by positive-sense RNA viruses. Over half of the FMA viral contigs were most similar to viruses reported from diverse hosts in aquatic environments, including phototrophs, invertebrates, and fungi. The detection of FMA viruses from orders dominated by plant viruses, namely Patatavirales and Tymovirales, indicate that members of these orders may thrive in aquatic hosts. PCR assays confirmed the presence of putative FMA plant viruses in asymptomatic vascular plants, indicating that viruses with persistent lifestyles are widespread in macrophytes. The detection of potato virus Y and oat blue dwarf virus in submerged macrophytes suggests that terrestrial plant viruses infect underwater plants and highlights a potential terrestrial-freshwater plant virus continuum. Defining the virome of unexplored macrophytes will improve our understanding of virus evolution in terrestrial and aquatic primary producers and reveal the potential ecological impacts of viral infection in macrophytes.

A World of Viruses Nested within Parasites: Unraveling Viral Diversity within Parasitic Flatworms (Platyhelminthes).

Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host shifts. And yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein, we uncovered viruses of flatworms (phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda, and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of neodermatans to a parasitic lifestyle. Most Neodermata viruses seem to codiversify with their host, with the exception of rhabdoviruses, which may switch hosts more often, based on phylogenetic relationships. Neodermata rhabdoviruses also have a position ancestral to vertebrate-associated rhabdo viruses, including lyssaviruses, suggesting that vertebrate-associated rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases. IMPORTANCE Little is known about the diversity of parasite-associated viruses and how these viruses may impact parasite fitness, parasite-host interactions, and virus evolution. The discovery of over a hundred viruses associated with a range of free-living and parasitic flatworms, including parasites of economic and clinical relevance, allowed us to compare the viromes of flatworms with contrasting lifestyles. The results suggest that flatworms acquired novel viruses after their transition to a parasitic lifestyle and highlight the possibility that they acquired viruses from their hosts and vice versa. An interesting example is the discovery of flatworm rhabdoviruses that have a position ancestral to rabies viruses and other vertebrate-associated rhabdoviruses, demonstrating that flatworm-associated viruses have emerged in a vertebrate host at least once in history. Therefore, parasitic flatworms may play a role in virus diversity and emergence. The roles that parasite-infecting viruses play in parasite-associated diseases remain to be investigated.

MIxS-SA: a MIxS extension defining the minimum information standard for sequence data from symbiont-associated micro-organisms.

The symbiont-associated (SA) environmental package is a new extension to the minimum information about any (x) sequence (MIxS) standards, established by the Parasite Microbiome Project (PMP) consortium, in collaboration with the Genomics Standard Consortium. The SA was built upon the host-associated MIxS standard, but reflects the nestedness of symbiont-associated microbiota within and across host-symbiont-microbe interactions. This package is designed to facilitate the collection and reporting of a broad range of metadata information that apply to symbionts such as life history traits, association with one or multiple host organisms, or the nature of host-symbiont interactions along the mutualism-parasitism continuum. To better reflect the inherent nestedness of all biological systems, we present a novel feature that allows users to co-localize samples, to nest a package within another package, and to identify replicates. Adoption of the MIxS-SA and of the new terms will facilitate reports of complex sampling design from a myriad of environments.

Active and Covert Infections of Cricket Iridovirus and Acheta domesticus Densovirus in Reared Gryllodes sigillatus Crickets.

Interest in developing food, feed, and other useful products from farmed insects has gained remarkable momentum in the past decade. Crickets are an especially popular group of farmed insects due to their nutritional quality, ease of rearing, and utility. However, production of crickets as an emerging commodity has been severely impacted by entomopathogenic infections, about which we know little. Here, we identified and characterized an unknown entomopathogen causing mass mortality in a lab-reared population of Gryllodes sigillatus crickets, a species used as an alternative to the popular Acheta domesticus due to its claimed tolerance to prevalent entomopathogenic viruses. Microdissection of sick and healthy crickets coupled with metagenomics-based identification and real-time qPCR viral quantification indicated high levels of cricket iridovirus (CrIV) in a symptomatic population, and evidence of covert CrIV infections in a healthy population. Our study also identified covert infections of Acheta domesticus densovirus (AdDNV) in both populations of G. sigillatus. These results add to the foundational research needed to better understand the pathology of mass-reared insects and ultimately develop the prevention, mitigation, and intervention strategies needed for economical production of insects as a commodity.

Spatial and Temporal Dynamics of Prokaryotic and Viral Community Assemblages in a Lotic System (Manatee Springs, Florida).

Flow from high-magnitude springs fed by the Floridan aquifer system contributes hundreds of liters of water per second to rivers, creating unique lotic systems. Despite their importance as freshwater sources and their contributions to the state's major rivers, little is known about the composition and spatiotemporal variability of prokaryotic and viral communities of these spring systems or their influence on downstream river sites. At four time points throughout a year, we determined the abundance and diversity of prokaryotic and viral communities at three sites within the first-magnitude Manatee Springs system (the spring head where water emerges from the aquifer, a mixed region where the spring run ends, and a downstream site in the Suwannee River). The abundance of prokaryotes and virus-like particles increased 100-fold from the spring head to the river and few members from the head communities persisted in the river at low abundance, suggesting the springs play a minor role in seeding downstream communities. Prokaryotic and viral communities within Manatee Springs clustered by site, with seasonal variability likely driven by flow. As water flowed through the system, microbial community composition was affected by changes in physiochemical parameters and community coalescence. Evidence of species sorting and mass effects could be seen in the assemblages. Greater temporal fluctuations were observed in prokaryotic and viral community composition with increasing distance from the spring outflow, reflecting the relative stability of the groundwater environment, and comparisons to springs from prior work reaffirmed that distinct first-magnitude springs support unique communities. IMPORTANCE Prokaryotic and viral communities are central to food webs and biogeochemical processes in aquatic environments, where they help maintain ecosystem health. The Floridan aquifer system (FAS), which is the primary drinking water source for millions of people in the southeastern United States, contributes large amounts of freshwater to major river systems in Florida through its springs. However, there is a paucity of information regarding the spatiotemporal dynamics of microbial communities in these essential flowing freshwater systems. This work explored the prokaryotic and viral communities in a first-magnitude spring system fed by the FAS that discharges millions of liters of water per day into the Suwannee River. This study examined microbial community composition through space and time as well as the environmental parameters and metacommunity assembly mechanisms that shape these communities, providing a foundational understanding for monitoring future changes.

Characterization of viruses in a tapeworm: phylogenetic position, vertical transmission, and transmission to the parasitized host.

Parasitic flatworms (Neodermata) infect all vertebrates and represent a significant health and economic burden worldwide due to the debilitating diseases they cause. This study sheds light for the first time into the virome of a tapeworm by describing six novel RNA virus candidate species associated with Schistocephalus solidus, including three negative-strand RNA viruses (order Jingchuvirales, Mononegavirales, and Bunyavirales) and three double-stranded RNA viruses. Using in vitro culture of S. solidus, controlled experimental infections and field sampling, we demonstrate that five of these viruses are vertically transmitted, and persist throughout the S. solidus complex life cycle. Moreover, we show that one of the viruses, named Schistocephalus solidus rhabdovirus (SsRV1), is excreted by the parasite and transmitted to parasitized hosts indicating that it may impact S. solidus-host interactions. In addition, SsRV1 has a basal phylogenetic position relative to vertebrate rhabdoviruses suggesting that parasitic flatworms could have contributed to virus emergence. Viruses similar to four of the S. solidus viruses identified here were found in geographically distant S. solidus populations through data mining. Further studies are necessary to determine if flatworm viruses can replicate in parasitized hosts, how they contribute to parasite infection dynamics and if these viruses could be targeted for treatment of parasitic disease.

Prokaryotic and Viral Community Composition of Freshwater Springs in Florida, USA.

Aquifers, which are essential underground freshwater reservoirs worldwide, are understudied ecosystems that harbor diverse forms of microbial life. This study investigated the abundance and composition of prokaryotic and viral communities in the outflow of five springs across northern Florida, USA, as a proxy of microbial communities found in one of the most productive aquifers in the world, the Floridan aquifer. The average abundances of virus-like particles and prokaryotic cells were slightly lower than those reported from other groundwater systems, ranging from 9.6 × 103 ml-1 to 1.1 × 105 ml-1 and 2.2 × 103 ml-1 to 3.4 × 104 ml-1, respectively. Despite all of the springs being fed by the Floridan aquifer, sequencing of 16S rRNA genes and viral metagenomes (viromes) revealed unique communities in each spring, suggesting that groundwater microbial communities are influenced by land usage in recharge zones. The prokaryotic communities were dominated by Bacteria, and though the most abundant phyla (Proteobacteria, Cyanobacteria, and Bacteroidetes) were found in relatively high abundance across springs, variation was seen at finer taxonomic resolution. The viral sequences were most similar to those described from other aquatic environments. Sequencing resulted in the completion of 58 novel viral genomes representing members of the order Caudovirales as well as prokaryotic and eukaryotic single-stranded DNA (ssDNA) viruses. Sequences similar to those of ssDNA viruses were detected at all spring sites and dominated the identifiable sequences at one spring site, showing that these small viruses merit further investigation in groundwater systems.IMPORTANCE Aquifer systems may hold up to 40% of the total microbial biomass on Earth. However, little is known about the composition of microbial communities within these critical freshwater ecosystems. Here, we took advantage of Florida's first-magnitude springs (the highest spring classification based on water discharge), each discharging at least 246 million liters of water each day from the Floridan aquifer system (FAS), to investigate prokaryotic and viral communities from the aquifer. The FAS serves as a major source of potable water in the Southeastern United States, providing water for large cities and citizens in three states. Unfortunately, the health of the FAS and its associated springs has declined in the past few decades due to nutrient loading, increased urbanization and agricultural activity in aquifer recharge zones, and saltwater intrusion. This is the first study to describe the prokaryotic and viral communities in Florida's first-magnitude springs, providing a baseline against which to compare future ecosystem change.

Prevalence of a vertically transmitted single-stranded DNA virus in spinybacked orbweavers (Gasteracantha cancriformis) from Florida, USA.

Spiders (order Araneae, class Arachnida) are an important group of predatory arthropods in terrestrial ecosystems that have been recently identified as an untapped reservoir of single-stranded (ss)DNA viruses. Specifically, spiders harbour a diversity of ssDNA viruses encoding a replication-associated protein (Rep) within a circular genome. However, little is known about the ecology of novel circular Rep-encoding ssDNA (CRESS DNA) viruses. Here we investigated two CRESS DNA viruses recently identified in spinybacked orbweavers (Gasteracantha cancriformis), namely spinybacked orbweaver circular virus (SpOrbCV) 1 and 2. SpOrbCV-1 was detected in the majority (> 65 %) of spider specimens from all life stages, including eggs, spiderlings and adults, demonstrating that this virus is active within spinybacked orbweavers. In contrast, SpOrbCV-2 was only detected in adults at a lower (36 %) prevalence. Since we also detected SpOrbCV-2 in other spider species and this virus has been reported from a dragonfly, we suggest that SpOrbCV-2 is accumulated in these predators through common insect prey. The prevalence of SpOrbCV-1 in collected specimens allowed us to design assays to characterize this virus, which represents a new group of CRESS DNA viruses, the 'circularisviruses'. To our knowledge, SpOrbCV-1 is the first example of a vertically transmitted virus in spiders, which may explain its high prevalence in spinybacked orbweavers. Since vertically transmitted viruses infecting insects (class Insecta) can manipulate their host's behaviour and physiology, future studies should investigate the ecological role of vertically transmitted viruses in spiders.

Novel Circular Rep-Encoding Single-Stranded DNA Viruses Detected in Treated Wastewater.

Here, we present the complete genome sequences of three circular replication-associated protein (Rep)-encoding single-stranded DNA (CRESS DNA) viruses detected in secondary treated and disinfected wastewater effluent. The discovered viruses, named wastewater CRESS DNA virus (WCDV)-1 to -3, represent novel viral species that seem to persist in wastewater effluent.

Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS DNA) Viruses: Ubiquitous Viruses With Small Genomes and a Diverse Host Range.

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.

Minimum Information about an Uncultivated Virus Genome (MIUViG).

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

Virus discovery in all three major lineages of terrestrial arthropods highlights the diversity of single-stranded DNA viruses associated with invertebrates.

Viruses encoding a replication-associated protein (Rep) within a covalently closed, single-stranded (ss)DNA genome are among the smallest viruses known to infect eukaryotic organisms, including economically valuable agricultural crops and livestock. Although circular Rep-encoding ssDNA (CRESS DNA) viruses are a widespread group for which our knowledge is rapidly expanding, biased sampling toward vertebrates and land plants has limited our understanding of their diversity and evolution. Here, we screened terrestrial arthropods for CRESS DNA viruses and report the identification of 44 viral genomes and replicons associated with specimens representing all three major terrestrial arthropod lineages, namely Euchelicerata (spiders), Hexapoda (insects), and Myriapoda (millipedes). We identified virus genomes belonging to three established CRESS DNA viral families (Circoviridae, Genomoviridae, and Smacoviridae); however, over half of the arthropod-associated viral genomes are only distantly related to currently classified CRESS DNA viral sequences. Although members of viral and satellite families known to infect plants (Geminiviridae, Nanoviridae, Alphasatellitidae) were not identified in this study, these plant-infecting CRESS DNA viruses and replicons are transmitted by hemipterans. Therefore, members from six out of the seven established CRESS DNA viral families circulate among arthropods. Furthermore, a phylogenetic analysis of Reps, including endogenous viral sequences, reported to date from a wide array of organisms revealed that most of the known CRESS DNA viral diversity circulates among invertebrates. Our results highlight the vast and unexplored diversity of CRESS DNA viruses among invertebrates and parallel findings from RNA viral discovery efforts in undersampled taxa.

Unprecedented Diversity of ssDNA Phages from the Family Microviridae Detected within the Gut of a Protochordate Model Organism (Ciona robusta).

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 Pequeñovirus) 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.

Discovery of Four Novel Circular Single-Stranded DNA Viruses in Fungus-Farming Termites.

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.