Single-cell RNA-seq of the rare virosphere reveals the native hosts of giant viruses in the marine environment.

Giant viruses (phylum Nucleocytoviricota) are globally distributed in aquatic ecosystems. They play fundamental roles as evolutionary drivers of eukaryotic plankton and regulators of global biogeochemical cycles. However, we lack knowledge about their native hosts, hindering our understanding of their life cycle and ecological importance. In the present study, we applied a single-cell RNA sequencing (scRNA-seq) approach to samples collected during an induced algal bloom, which enabled pairing active giant viruses with their native protist hosts. We detected hundreds of single cells from multiple host lineages infected by diverse giant viruses. These host cells included members of the algal groups Chrysophycae and Prymnesiophycae, as well as heterotrophic flagellates in the class Katablepharidaceae. Katablepharids were infected with a rare Imitervirales-07 giant virus lineage expressing a large repertoire of cell-fate regulation genes. Analysis of the temporal dynamics of these host-virus interactions revealed an important role for the Imitervirales-07 in controlling the population size of the host Katablepharid population. Our results demonstrate that scRNA-seq can be used to identify previously undescribed host-virus interactions and study their ecological importance and impact.

Mirusviruses link herpesviruses to giant viruses.

DNA viruses have a major influence on the ecology and evolution of cellular organisms1-4, but their overall diversity and evolutionary trajectories remain elusive5. Here we carried out a phylogeny-guided genome-resolved metagenomic survey of the sunlit oceans and discovered plankton-infecting relatives of herpesviruses that form a putative new phylum dubbed Mirusviricota. The virion morphogenesis module of this large monophyletic clade is typical of viruses from the realm Duplodnaviria6, with multiple components strongly indicating a common ancestry with animal-infecting Herpesvirales. Yet, a substantial fraction of mirusvirus genes, including hallmark transcription machinery genes missing in herpesviruses, are closely related homologues of giant eukaryotic DNA viruses from another viral realm, Varidnaviria. These remarkable chimaeric attributes connecting Mirusviricota to herpesviruses and giant eukaryotic viruses are supported by more than 100 environmental mirusvirus genomes, including a near-complete contiguous genome of 432 kilobases. Moreover, mirusviruses are among the most abundant and active eukaryotic viruses characterized in the sunlit oceans, encoding a diverse array of functions used during the infection of microbial eukaryotes from pole to pole. The prevalence, functional activity, diversification and atypical chimaeric attributes of mirusviruses point to a lasting role of Mirusviricota in the ecology of marine ecosystems and in the evolution of eukaryotic DNA viruses.

Aquatic Flaviviruses.

Flaviviruses are positive-sense single-stranded RNA viruses, including some well-known human pathogens such as Zika, dengue, and yellow fever viruses, which are primarily associated with mosquito and tick vectors. The vast majority of flavivirus research has focused on terrestrial environments; however, recent findings indicate that a range of flaviviruses are also present in aquatic environments, both marine and freshwater. These flaviviruses are found in various hosts, including fish, crustaceans, molluscs, and echinoderms. Although the effects of aquatic flaviviruses on the hosts they infect are not all known, some have been detected in farmed species and may have detrimental effects on the aquaculture industry. Exploration of the evolutionary history through the discovery of the Wenzhou shark flavivirus in both a shark and crab host is of particular interest since the potential dual-host nature of this virus may indicate that the invertebrate-vertebrate relationship seen in other flaviviruses may have a more profound evolutionary root than previously expected. Potential endogenous viral elements and the range of novel aquatic flaviviruses discovered thus shed light on virus origins and evolutionary history and may indicate that, like terrestrial life, the origins of flaviviruses may lie in aquatic environments.

Viromes in marine ecosystems reveal remarkable invertebrate RNA virus diversity.

Little is known about ocean viromes and the ecological drivers of the evolution of aquatic RNA viruses. This study employed a meta-transcriptomic approach to characterize the viromes of 58 marine invertebrate species across three seas. This revealed the presence of 315 newly identified RNA viruses in nine viral families or orders (Durnavirales, Totiviridae, Bunyavirales, Hantaviridae, Picornavirales, Flaviviridae, Hepelivirales, Solemoviridae, and Tombusviridae), with most of them being sufficiently divergent to the already documented viruses. Notably, this study revealed three marine invertebrate hantaviruses that are rooted to vertebrate hantaviruses, further supporting that hantaviruses may have a marine origin. We have also found evidence for possible host sharing and switch events during virus evolution. Overall, we have revealed the hidden diversity of marine invertebrate RNA viruses.

Climate change: how it impacts the emergence, transmission, resistance and consequences of viral infections in animals and plants.

The ongoing COVID-19 pandemic has made us wonder what led to its occurrence and what can be done to avoid such events in the future. As we document, one changing circumstance that is resulting in the emergence and changing the expression of viral diseases in both plants and animals is climate change. Of note, the rapidly changing environment and weather conditions such as excessive flooding, droughts, and forest fires have raised concerns about the global ecosystem's security, sustainability, and balance. In this review, we discuss the main consequences of climate change and link these to how they impact the appearance of new viral pathogens, how they may facilitate transmission between usual and novel hosts, and how they may also affect the host's ability to manage the infection. We emphasize how changes in temperature and humidity and other events associated with climate change influence the reservoirs of viral infections, their transmission by insects and other intermediates, their survival outside the host as well the success of infection in plants and animals. We conclude that climate change has mainly detrimental consequences for the emergence, transmission, and outcome of viral infections and plead the case for halting and hopefully reversing this dangerous event.

Polinton-like viruses are abundant in aquatic ecosystems.

BACKGROUND: Polintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited. RESULTS: Here, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae. CONCLUSIONS: Our study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups. Video abstract.

Current progress on identification of virus pathogens and the antiviral effectors in echinoderms.

Echinoderms are important marine organisms that live in a wide range from the intertidal zone to the abyssal zone. Members of this phylum are prone to dramatic population fluctuations that may trigger dramatic shifts in ecosystem structure. Despite the extremely complex nature of the marine environment, the immune systems of echinoderms induce a complex innate immune response to prokaryotic and eukaryotic pathogens. Previous studies showed that many echinoderm disease outbreaks were associated with specific bacteria, whereas recent scientific investigations using newly developed technologies revealed the amazing diversity of viruses in seawater. Viruses are potential pathogens of several infectious diseases of marine echinoderms. We reviewed the discovery of viruses in echinoderms and discussed the relationship between viruses and diseases for the first time. We further summarized the research progress of the potential immune-related genes and signal pathways induced by viruses and poly (I:C). Additionally, numbers of studies showed that active substances extracted from echinoderms, or the compounds synthesized from these substances, have significant antihuman virus ability. This result suggests that the active substances derived from echinoderms provide potential antiviral protection for the organism, which may provide future research directions for the antiviral immunity of echinoderms. Thus, this review also collected information on the antiviral activities of biologically active substances from echinoderms, which may pave the way for new trends in antiviral immunity for echinoderms and antiviral drugs in humans.

Significance of Viral Activity for Regulating Heterotrophic Prokaryote Community Dynamics along a Meridional Gradient of Stratification in the Northeast Atlantic Ocean.

How microbial populations interact influences the availability and flux of organic carbon in the ocean. Understanding how these interactions vary over broad spatial scales is therefore a fundamental aim of microbial oceanography. In this study, we assessed variations in the abundances, production, virus and grazing induced mortality of heterotrophic prokaryotes during summer along a meridional gradient in stratification in the North Atlantic Ocean. Heterotrophic prokaryote abundance and activity varied with phytoplankton biomass, while the relative distribution of prokaryotic subpopulations (ratio of high nucleic acid fluorescent (HNA) and low nucleic acid fluorescent (LNA) cells) was significantly correlated to phytoplankton mortality mode (i.e., viral lysis to grazing rate ratio). Virus-mediate morality was the primary loss process regulating the heterotrophic prokaryotic communities (average 55% of the total mortality), which may be attributed to the strong top-down regulation of the bacterivorous protozoans. Host availability, encounter rate, and HNA:LNA were important factors regulating viral dynamics. Conversely, the abundance and activity of bacterivorous protozoans were largely regulated by temperature and turbulence. The ratio of total microbial mediated mortality to total available prokaryote carbon reveals that over the latitudinal gradient the heterotrophic prokaryote community gradually moved from a near steady state system regulated by high turnover in subtropical region to net heterotrophic production in the temperate region.

Seasonal and diel patterns of abundance and activity of viruses in the Red Sea.

Virus-microbe interactions have been studied in great molecular details for many years in cultured model systems, yielding a plethora of knowledge on how viruses use and manipulate host machinery. Since the advent of molecular techniques and high-throughput sequencing, methods such as cooccurrence, nucleotide composition, and other statistical frameworks have been widely used to infer virus-microbe interactions, overcoming the limitations of culturing methods. However, their accuracy and relevance is still debatable as cooccurrence does not necessarily mean interaction. Here we introduce an ecological perspective of marine viral communities and potential interaction with their hosts, using analyses that make no prior assumptions on specific virus-host pairs. By size fractionating water samples into free viruses and microbes (i.e., also viruses inside or attached to their hosts) and looking at how viral group abundance changes over time along both fractions, we show that the viral community is undergoing a change in rank abundance across seasons, suggesting a seasonal succession of viruses in the Red Sea. We use abundance patterns in the different size fractions to classify viral clusters, indicating potential diverse interactions with their hosts and potential differences in life history traits between major viral groups. Finally, we show hourly resolved variations of intracellular abundance of similar viral groups, which might indicate differences in their infection cycles or metabolic capacities.

Influence of the polar light cycle on seasonal dynamics of an Antarctic lake microbial community.

BACKGROUND: Cold environments dominate the Earth's biosphere and microbial activity drives ecosystem processes thereby contributing greatly to global biogeochemical cycles. Polar environments differ to all other cold environments by experiencing 24-h sunlight in summer and no sunlight in winter. The Vestfold Hills in East Antarctica contains hundreds of lakes that have evolved from a marine origin only 3000-7000 years ago. Ace Lake is a meromictic (stratified) lake from this region that has been intensively studied since the 1970s. Here, a total of 120 metagenomes representing a seasonal cycle and four summers spanning a 10-year period were analyzed to determine the effects of the polar light cycle on microbial-driven nutrient cycles. RESULTS: The lake system is characterized by complex sulfur and hydrogen cycling, especially in the anoxic layers, with multiple mechanisms for the breakdown of biopolymers present throughout the water column. The two most abundant taxa are phototrophs (green sulfur bacteria and cyanobacteria) that are highly influenced by the seasonal availability of sunlight. The extent of the Chlorobium biomass thriving at the interface in summer was captured in underwater video footage. The Chlorobium abundance dropped from up to 83% in summer to 6% in winter and 1% in spring, before rebounding to high levels. Predicted Chlorobium viruses and cyanophage were also abundant, but their levels did not negatively correlate with their hosts. CONCLUSION: Over-wintering expeditions in Antarctica are logistically challenging, meaning insight into winter processes has been inferred from limited data. Here, we found that in contrast to chemolithoautotrophic carbon fixation potential of Southern Ocean Thaumarchaeota, this marine-derived lake evolved a reliance on photosynthesis. While viruses associated with phototrophs also have high seasonal abundance, the negative impact of viral infection on host growth appeared to be limited. The microbial community as a whole appears to have developed a capacity to generate biomass and remineralize nutrients, sufficient to sustain itself between two rounds of sunlight-driven summer-activity. In addition, this unique metagenome dataset provides considerable opportunity for future interrogation of eukaryotes and their viruses, abundant uncharacterized taxa (i.e. dark matter), and for testing hypotheses about endemic species in polar aquatic ecosystems. Video Abstract.

Temporal transcriptional patterns of cyanophage genes suggest synchronized infection of cyanobacteria in the oceans.

BACKGROUND: Based on the peak expression times during infection, early, middle, and late genes have been characterized in viruses (cyanophages) that infect the unicellular cyanobacterium Prochlorococcus. Laboratory experiments show that some cyanophages can only replicate in the light and thus exhibit diurnal infection rhythms under light-dark cycles. Field evidence also suggests synchronized infection of Prochlorococcus by cyanophages in the oceans, which should result in progressive expression of cyanophage early, middle, and late genes. However, distinct temporal expression patterns have not been observed in cyanophage field populations. RESULTS: In this study, we reanalyzed a previous metatranscriptomic dataset collected in the North Pacific Subtropical Gyre. In this dataset, it was previously shown that aggregate transcripts from cyanophage scaffolds display diurnal transcriptional rhythms with transcript abundances decreasing at night. By mapping metatranscriptomic reads to individual viral genes, we identified periodically expressed genes from putative viruses infecting the cyanobacteria Prochlorococcus and Synechococcus, heterotrophic bacteria, and algae. Of the 41 cyanophage genes, 35 were from cyanomyoviruses. We grouped the periodically expressed cyanomyovirus genes into early, middle, and late genes based on the conserved temporal expression patterns of their orthologs in cyanomyovirus laboratory cultures. We found that the peak expression times of late genes in cyanophage field populations were significantly later than those of early and middle genes, which were similar to the temporal expression patterns of synchronized cyanophage laboratory cultures. CONCLUSIONS: The significantly later peak expression times of late genes in cyanomyovirus field populations suggest that cyanophage infection of Prochlorococcus is synchronized in the North Pacific Subtropical Gyre. The night-time peak expression of late genes also suggests synchronized lysis of Prochlorococcus at night, which might result in synchronized release of dissolved organic matter to the marine food web. Video abstract.

Diseases and pathogens of marine invertebrate corals in Indian reefs.

Diseases in marine invertebrate corals have been reported worldwide and have been associated with infection by various microbial pathogens that cause massive mortality. Several bacterial species, especially Vibrio species but also members of the cyanobacteria, fungi, viruses, and protists, are described as important pathogens associated with coral disease and mortality. The present work provides an updated overview of main diseases and implicated microbial species affecting corals in Indian reefs. Further study on pathogen diversity, classification, spread and environmental factors on pathogen-host interactions may contribute a better understanding of the coral diseases.

Marine virus predation by non-host organisms.

Viruses are the most abundant biological entities in marine environments, however, despite its potential ecological implications, little is known about virus removal by ambient non-host organisms. Here, we examined the effects of a variety of non-host organisms on the removal of viruses. The marine algal virus PgV-07T (infective to Phaeocystis globosa) can be discriminated from bacteriophages using flow cytometry, facilitating its use as a representative model system. Of all the non-host organisms tested, anemones, polychaete larvae, sea squirts, crabs, cockles, oysters and sponges significantly reduced viral abundance. The latter four species reduced viral abundance the most, by 90, 43, 12 and 98% over 24 h, respectively. Breadcrumb sponges instantly removed viruses at high rates (176 mL h-1 g tissue dry wt-1) which continued over an extended period of time. The variety of non-host organisms capable of reducing viral abundance highlights that viral loss by ambient organisms is an overlooked avenue of viral ecology. Moreover, our finding that temperate sponges have the huge potential for constant and effective removal of viruses from the water column demonstrates that natural viral loss has, thus far, been underestimated.

A Brief Introduction to Influenza A Virus in Marine Mammals.

Influenza A infection has been detected in marine mammals going back to 1975, with additional unconfirmed outbreaks as far back as 1931. Over the past forty years, infectious virus has been recovered on ten separate occasions from both pinnipeds (harbor seal, elephant seal, and Caspian seal) and cetaceans (striped whale and pilot whale). Recovered viruses have spanned a range of subtypes (H1, H3, H4, H7, H10, and H13) and, in all but H1N1, show strong evidence for deriving directly from avian sources. To date, there have been five unusual mortality events directly attributed to influenza A virus; these have primarily occurred in harbor seals in the Northeastern United States, with the most recent occurring in harbor seals in the North Sea.There are numerous additional reports wherein influenza A virus has indirectly been identified in marine mammals; these include serosurveillance efforts that have detected influenza A- and B-specific antibodies in marine mammals spanning the globe and the detection of viral RNA in both active and opportunistic surveillance in the Northwest Atlantic. For viral detection and recovery, nasal, rectal, and conjunctival swabs have been employed in pinnipeds, while blowhole, nasal, and rectal swabs have been employed in cetaceans. In the case of deceased animals, virus has also been detected in tissue. Surveillance has historically been somewhat limited, relying largely upon opportunistic sampling of stranded or bycaught animals and primarily occurring in response to a mortality event. A handful of active surveillance projects have shown that influenza may be more endemic in marine mammals than previously appreciated, though live virus is difficult to recover. Surveillance efforts are hindered by permitting and logistical challenges, the absence of reagents and methodology optimized for nonavian wild hosts, and low concentration of virus recovered from asymptomatic animals. Despite these challenges, a growing body of evidence suggests that marine mammals are an important wild reservoir of influenza and may contribute to mammalian adaptation of avian variants.

dsRNA-seq Reveals Novel RNA Virus and Virus-Like Putative Complete Genome Sequences from Hymeniacidon sp. Sponge.

Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellular animal that belongs to the phylum Porifera, the marine sponge is one of the well-studied holobiont systems. In the present study, we elucidated the putative complete genome sequences of five novel RNA viruses from Hymeniacidon sponge using a combination of double-stranded RNA sequencing, called fragmented and primer ligated dsRNA sequencing, and a conventional transcriptome method targeting single-stranded RNA. We identified highly diverged RNA-dependent RNA polymerase sequences, including a potential novel RNA viral lineage, in the sponge and three viruses presumed to infect sponge cells.

Modeling Pathogen Dispersal in Marine Fish and Shellfish.

In marine ecosystems, oceanographic processes often govern host contacts with infectious agents. Consequently, many approaches developed to quantify pathogen dispersal in terrestrial ecosystems have limited use in the marine context. Recent applications in marine disease modeling demonstrate that physical oceanographic models coupled with biological models of infectious agents can characterize dispersal networks of pathogens in marine ecosystems. Biophysical modeling has been used over the past two decades to model larval dispersion but has only recently been utilized in marine epidemiology. In this review, we describe how biophysical models function and how they can be used to measure connectivity of infectious agents between sites, test hypotheses regarding pathogen dispersal, and quantify patterns of pathogen spread, focusing on fish and shellfish pathogens.

Molecular evidence for horizontal transmission of chelonid alphaherpesvirus 5 at green turtle (Chelonia mydas) foraging grounds in Queensland, Australia.

Fibropapillomatosis (FP) is a marine turtle disease recognised by benign tumours on the skin, eyes, shell, oral cavity and/or viscera. Despite being a globally distributed disease that affects an endangered species, research on FP and its likely causative agent chelonid alphaherpesvirus 5 (ChHV5) in Australia is limited. Here we present improved molecular assays developed for detection of ChHV5, in combination with a robust molecular and phylogenetic analysis of ChHV5 variants. This approach utilised a multi-gene assay to detect ChHV5 in all FP tumors sampled from 62 marine turtles found at six foraging grounds along the Great Barrier Reef. Six distinct variants of ChHV5 were identified and the distribution of these variants was associated with host foraging ground. Conversely, no association between host genetic origin and ChHV5 viral variant was found. Together this evidence supports the hypothesis that marine turtles undergo horizontal transmission of ChHV5 at foraging grounds and are unlikely to be contracting the disease at rookeries, either during mating or vertically from parent to offspring.

Long-term stability and Red Queen-like strain dynamics in marine viruses.

Viruses that infect microorganisms dominate marine microbial communities numerically, with impacts ranging from host evolution to global biogeochemical cycles1,2. However, virus community dynamics, necessary for conceptual and mechanistic model development, remains difficult to assess. Here, we describe the long-term stability of a viral community by analysing the metagenomes of near-surface 0.02-0.2 µm samples from the San Pedro Ocean Time-series3 that were sampled monthly over 5 years. Of 19,907 assembled viral contigs (>5 kb, mean 15 kb), 97% were found in each sample (by >98% ID metagenomic read recruitment) to have relative abundances that ranged over seven orders of magnitude, with limited temporal reordering of rank abundances along with little change in richness. Seasonal variations in viral community composition were superimposed on the overall stability; maximum community similarity occurred at 12-month intervals. Despite the stability of viral genotypic clusters that had 98% sequence identity, viral sequences showed transient variations in single-nucleotide polymorphisms (SNPs) and constant turnover of minor population variants, each rising and falling over a few months, reminiscent of Red Queen dynamics4. The rise and fall of variants within populations, interpreted through the perspective of known virus-host interactions5, is consistent with the hypothesis that fluctuating selection acts on a microdiverse cloud of strains, and this succession is associated with ever-shifting virus-host defences and counterdefences. This results in long-term virus-host coexistence that is facilitated by perpetually changing minor variants.

Metabolic and biogeochemical consequences of viral infection in aquatic ecosystems.

Ecosystems are controlled by 'bottom-up' (resources) and 'top-down' (predation) forces. Viral infection is now recognized as a ubiquitous top-down control of microbial growth across ecosystems but, at the same time, cell death by viral predation influences, and is influenced by, resource availability. In this Review, we discuss recent advances in understanding the biogeochemical impact of viruses, focusing on how metabolic reprogramming of host cells during lytic viral infection alters the flow of energy and nutrients in aquatic ecosystems. Our synthesis revealed several emerging themes. First, viral infection transforms host metabolism, in part through virus-encoded metabolic genes; the functions performed by these genes appear to alleviate energetic and biosynthetic bottlenecks to viral production. Second, viral infection depends on the physiological state of the host cell and on environmental conditions, which are challenging to replicate in the laboratory. Last, metabolic reprogramming of infected cells and viral lysis alter nutrient cycling and carbon export in the oceans, although the net impacts remain uncertain. This Review highlights the need for understanding viral infection dynamics in realistic physiological and environmental contexts to better predict their biogeochemical consequences.

A universal random DNA amplification and labeling strategy for microarray to detect multiple pathogens of aquatic animals.

Diseases caused by bacteria, fungi, and viruses pose a great threat to aquaculture. As DNA microarrays can be used to detect multiple pathogens, here we reported an array with the potential to simultaneously detect 13 bacterial and 11 viral pathogens of aquatic animals. The array included 853 oligonucleotide probes (20-40 mer) complementary to various virus-specific sequences and four chromosomal loci (16S rRNA, gyrB, dnaJ, and recA) of bacteria. Multiplex PCR, phi29 DNA polymerase, and a Klenow fragment-based method were evaluated for amplifying and labeling the nucleic acid of pathogens. While array hybridization signals were most intense using pathogen sequences amplified by multiplex PCR, the phi29 DNA polymerase method was more convenient and ideal since it did not require sequence-specific primers that could bias against detecting novel pathogens. The feasibility of the phi29 DNA polymerase-based microarray strategy was also demonstrated by detecting multiple unknown pathogens from four samples of diseased fish and shrimps.