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.

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology.

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.

Unmasking cellular response of a bloom-forming alga to viral infection by resolving expression profiles at a single-cell level.

Infection by large dsDNA viruses can lead to a profound alteration of host transcriptome and metabolome in order to provide essential building blocks to support the high metabolic demand for viral assembly and egress. Host response to viral infection can typically lead to diverse phenotypic outcome that include shift in host life cycle and activation of anti-viral defense response. Nevertheless, there is a major bottleneck to discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidic setup that captures individual algal cells and multiplex quantitate PCR. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and susceptible cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.

Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host.

Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilis NC64A host, and found that virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9-30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts.

Sterol and Sphingoid Glycoconjugates from Microalgae.

Microalgae are well known as primary producers in the hydrosphere. As sources of natural products, microalgae are attracting major attention due to the potential of their practical applications as valuable food constituents, raw material for biofuels, drug candidates, and components of drug delivery systems. This paper presents a short review of a low-molecular-weight steroid and sphingolipid glycoconjugates, with an analysis of the literature on their structures, functions, and bioactivities. The discussed data on sterols and the corresponding glycoconjugates not only demonstrate their structural diversity and properties, but also allow for a better understanding of steroid biogenesis in some echinoderms, mollusks, and other invertebrates which receive these substances from food and possibly from their microalgal symbionts. In another part of this review, the structures and biological functions of sphingolipid glycoconjugates are discussed. Their role in limiting microalgal blooms as a result of viral infections is emphasized.

Rapidity of Genomic Adaptations to Prasinovirus Infection in a Marine Microalga.

Prasinoviruses are large dsDNA viruses commonly found in aquatic systems worldwide, where they can infect and lyse unicellular prasinophyte algae such as Ostreococcus. Host susceptibility is virus strain-specific, but resistance of susceptible Ostreococcus tauri strains to a virulent virus arises frequently. In clonal resistant lines that re-grow, viruses are usually present for many generations, and genes clustered on chromosome 19 show physical rearrangements and differential expression. Here, we investigated changes occurring during the first two weeks after inoculation of the prasinovirus OtV5. By serial dilutions of cultures at the time of inoculation, we estimated the frequency of resistant cells arising in virus-challenged O. tauri cultures to be 10-3⁻10-4 of the inoculated population. Re-growing resistant cells were detectable by flow cytometry 3 days post-inoculation (dpi), visible re-greening of cultures occurred by 6 dpi, and karyotypic changes were visually detectable at 8 dpi. Resistant cell lines showed a modified spectrum of host-virus specificities and much lower levels of OtV5 adsorption.

Prasinovirus Attack of Ostreococcus Is Furtive by Day but Savage by Night.

Prasinoviruses are large DNA viruses that infect diverse genera of green microalgae worldwide in aquatic ecosystems, but molecular knowledge of their life cycles is lacking. Several complete genomes of both these viruses and their marine algal hosts are now available and have been used to show the pervasive presence of these species in microbial metagenomes. We have analyzed the life cycle of Ostreococcus tauri virus 5 (OtV5), a lytic virus, using transcriptome sequencing (RNA-Seq) from 12 time points of healthy or infected Ostreococcus tauri cells over a day/night cycle in culture. In the day, viral gene transcription remained low while host nitrogen metabolism gene transcription was initially strongly repressed for two successive time points before being induced for 8 h, but during the night, viral transcription increased steeply while host nitrogen metabolism genes were repressed and many host functions that are normally reduced in the dark appeared to be compensated either by genes expressed from the virus or by increased expression of a subset of 4.4% of the host's genes. Some host cells underwent lysis progressively during the night, but a larger proportion were lysed the following morning. Our data suggest that the life cycles of algal viruses mirror the diurnal rhythms of their hosts.IMPORTANCE Prasinoviruses are common in marine environments, and although several complete genomes of these viruses and their hosts have been characterized, little is known about their life cycles. Here we analyze in detail the transcriptional changes occurring over a 27-h-long experiment in a natural diurnal rhythm, in which the growth of host cells is to some extent synchronized, so that host DNA replication occurs late in the day or early in the night and cell division occurs during the night. Surprisingly, viral transcription remains quiescent over the daytime, when the most energy (from light) is available, but during the night viral transcription activates, accompanied by expression of a few host genes that are probably required by the virus. Although our experiment was accomplished in the lab, cyclical changes have been documented in host transcription in the ocean. Our observations may thus be relevant for eukaryotic phytoplankton in natural environments.

Morphological switch to a resistant subpopulation in response to viral infection in the bloom-forming coccolithophore Emiliania huxleyi.

Recognizing the life cycle of an organism is key to understanding its biology and ecological impact. Emiliania huxleyi is a cosmopolitan marine microalga, which displays a poorly understood biphasic sexual life cycle comprised of a calcified diploid phase and a morphologically distinct biflagellate haploid phase. Diploid cells (2N) form large-scale blooms in the oceans, which are routinely terminated by specific lytic viruses (EhV). In contrast, haploid cells (1N) are resistant to EhV. Further evidence indicates that 1N cells may be produced during viral infection. A shift in morphology, driven by meiosis, could therefore constitute a mechanism for E. huxleyi cells to escape from EhV during blooms. This process has been metaphorically coined the 'Cheshire Cat' (CC) strategy. We tested this model in two E. huxleyi strains using a detailed assessment of morphological and ploidy-level variations as well as expression of gene markers for meiosis and the flagellate phenotype. We showed that following the CC model, production of resistant cells was triggered during infection. This led to the rise of a new subpopulation of cells in the two strains that morphologically resembled haploid cells and were resistant to EhV. However, ploidy-level analyses indicated that the new resistant cells were diploid or aneuploid. Thus, the CC strategy in E. huxleyi appears to be a life-phase switch mechanism involving morphological remodeling that is decoupled from meiosis. Our results highlight the adaptive significance of morphological plasticity mediating complex host-virus interactions in marine phytoplankton.

Communication via extracellular vesicles enhances viral infection of a cosmopolitan alga.

Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure 1 . Extracellular vesicles (EVs) are produced by bacteria 2,3 , archaea 4 , protists 5 and metazoans, and can mediate pathogenicity 6 or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment 7 . Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae) 8 , which leads to the demise of these large-scale oceanic blooms 9,10 . We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients 11,12 , this mode of cell-cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.

Biotic interactions as drivers of algal origin and evolution.

Contents 670 I. 671 II. 671 III. 676 IV. 678 678 References 678 SUMMARY: Biotic interactions underlie life's diversity and are the lynchpin to understanding its complexity and resilience within an ecological niche. Algal biologists have embraced this paradigm, and studies building on the explosive growth in omics and cell biology methods have facilitated the in-depth analysis of nonmodel organisms and communities from a variety of ecosystems. In turn, these advances have enabled a major revision of our understanding of the origin and evolution of photosynthesis in eukaryotes, bacterial-algal interactions, control of massive algal blooms in the ocean, and the maintenance and degradation of coral reefs. Here, we review some of the most exciting developments in the field of algal biotic interactions and identify challenges for scientists in the coming years. We foresee the development of an algal knowledgebase that integrates ecosystem-wide omics data and the development of molecular tools/resources to perform functional analyses of individuals in isolation and in populations. These assets will allow us to move beyond mechanistic studies of a single species towards understanding the interactions amongst algae and other organisms in both the laboratory and the field.

Variation in the Genetic Repertoire of Viruses Infecting Micromonas pusilla Reflects Horizontal Gene Transfer and Links to Their Environmental Distribution.

Prasinophytes, a group of eukaryotic phytoplankton, has a global distribution and is infected by large double-stranded DNA viruses (prasinoviruses) in the family Phycodnaviridae. This study examines the genetic repertoire, phylogeny, and environmental distribution of phycodnaviruses infecting Micromonas pusilla, other prasinophytes and chlorophytes. Based on comparisons among the genomes of viruses infecting M. pusilla and other phycodnaviruses, as well as the genome from a host isolate of M. pusilla, viruses infecting M. pusilla (MpVs) share a limited set of core genes, but vary strongly in their flexible pan-genome that includes numerous metabolic genes, such as those associated with amino acid synthesis and sugar manipulation. Surprisingly, few of these presumably host-derived genes are shared with M. pusilla, but rather have their closest non-viral homologue in bacteria and other eukaryotes, indicating horizontal gene transfer. A comparative analysis of full-length DNA polymerase (DNApol) genes from prasinoviruses with their overall gene content, demonstrated that the phylogeny of DNApol gene fragments reflects the gene content of the viruses; hence, environmental DNApol gene sequences from prasinoviruses can be used to infer their overall genetic repertoire. Thus, the distribution of virus ecotypes across environmental samples based on DNApol sequences implies substantial underlying differences in gene content that reflect local environmental conditions. Moreover, the high diversity observed in the genetic repertoire of prasinoviruses has been driven by horizontal gene transfer throughout their evolutionary history, resulting in a broad suite of functional capabilities and a high diversity of prasinovirus ecotypes.

Effect of Chlorovirus ATCV-1 infection on behavior of C57Bl/6 mice.

Neuroinflammation induced during immune responses to viral infections in the brain affect behavior. Unexpected evidence that oral gavage of an algal virus in its host algal cells could alter cognition was further examined by directly injecting purified algal virus ATCV-1 intracranially into C57BL/6 mice. After 4weeks, the ATCV-1 infection impaired delayed location recognition memory, and also reduced and anxiety. Corresponding to these effects, heightened ATCV-1, IL-6, iNOS, IFN-γ, and CD11b expression in brains was observed 3-days and/or 8-weeks post infection compared with control mice. These results imply that ATCV-1 infection damages the hippocampus via induction of inflammatory factors.

Infection of phytoplankton by aerosolized marine viruses.

Marine viruses constitute a major ecological and evolutionary driving force in the marine ecosystems. However, their dispersal mechanisms remain underexplored. Here we follow the dynamics of Emiliania huxleyi viruses (EhV) that infect the ubiquitous, bloom-forming phytoplankton E. huxleyi and show that EhV are emitted to the atmosphere as primary marine aerosols. Using a laboratory-based setup, we showed that the dynamic of EhV aerial emission is strongly coupled to the host-virus dynamic in the culture media. In addition, we recovered EhV DNA from atmospheric samples collected over an E. huxleyi bloom in the North Atlantic, providing evidence for aerosolization of marine viruses in their natural environment. Decay rate analysis in the laboratory revealed that aerosolized viruses can remain infective under meteorological conditions prevailing during E. huxleyi blooms in the ocean, allowing potential dispersal and infectivity over hundreds of kilometers. Based on the combined laboratory and in situ findings, we propose that atmospheric transport of EhV is an effective transmission mechanism for spreading viral infection over large areas in the ocean. This transmission mechanism may also have an important ecological impact on the large-scale host-virus "arms race" during bloom succession and consequently the turnover of carbon in the ocean.

Marine Viruses that infect Eukaryotic Microalgae.

Marine microalgae, in general, explain large amount of the primary productions on the planet. Their huge biomass through photosynthetic activities is significant to understand the global geochemical cycles. Many researchers are, therefore, focused on studies of marine microalgae, i.e. phytoplankton. Since the first report of high abundance of viruses in the sea at late 1980's, the marine viruses have recognized as an important decreasing factor of its host populations. They seem to be composed of diverse viruses infectious to different organism groups; most of them are considered to be phages infectious to prokaryotes, and viruses infecting microalgae might be ranked in second level. Over the last quarter of a century, the knowledge on marine microalgal viruses has been accumulated in many aspects. Until today, ca. 40 species of marine microalgal viruses have been discovered, including dsDNA, ssDNA, dsRNA and ssRNA viruses. Their features are unique and comprise new ideas and discoveries, indicating that the marine microalgal virus research is still an intriguing unexplored field. In this review, we summarize their basic biology and ecology, and discuss how and what we should research in this area for further progress.

Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host-virus interactions.

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins.

Novel molecular determinants of viral susceptibility and resistance in the lipidome of Emiliania huxleyi.

Viruses play a key role in controlling the population dynamics of algae, including Emiliania huxleyi, a globally distributed haptophyte with calcite coccoliths that comprise ca. 50% of the sinking carbonate flux from the surface ocean. Emiliania huxleyi viruses (EhVs) routinely infect and terminate E. huxleyi blooms. EhVs are surrounded by a lipid envelope, which we found to be comprised largely of glycosphingolipids (GSLs) with lesser amounts of polar glycerolipids. Infection appears to involve membrane fusion between the virus and host, and we hypothesized that specific polar lipids may facilitate virus attachment. We identified three novel intact polar lipids in E. huxleyi strain CCMP 374 and EhV86, including a GSL with a monosaccharide sialic acid headgroup (sGSL); for all 11 E. huxleyi strains we tested, there was a direct relationship between sGSL content and sensitivity to infection by EhV1, EhV86 and EhV163. In mesocosms, the E. huxleyi population with greatest initial sGSL content had the highest rate of virus-induced mortality. We propose potential physiological roles for sGSL that would be beneficial for growth but leave cells susceptible to infection, thus furthering the discussion of Red Queen-based co-evolution and the cost(s) of sensitivity and resistance in the dynamic E. huxleyi-EhV system.

Unveiling the transcriptional features associated with coccolithovirus infection of natural Emiliania huxleyi blooms.

Lytic viruses have been implicated in the massive cellular lysis observed during algal blooms, through which they assume a prominent role in oceanic carbon and nutrient flows. Despite their impact on biogeochemical cycling, the transcriptional dynamics of these important oceanic events is still poorly understood. Here, we employ an oligonucleotide microarray to monitor host (Emiliania huxleyi) and virus (coccolithovirus) transcriptomic features during the course of E. huxleyi blooms induced in seawater-based mesocosm enclosures. Host bloom development and subsequent coccolithovirus infection was associated with a major shift in transcriptional profile. In addition to the expected metabolic requirements typically associated with viral infection (amino acid and nucleotide metabolism, as well as transcription- and replication-associated functions), the results strongly suggest that the manipulation of lipid metabolism plays a fundamental role during host-virus interaction. The results herein reveal the scale, so far massively underestimated, of the transcriptional domination that occurs during coccolithovirus infection in the natural environment.

A chemical arms race at sea mediates algal host-virus interactions.

Despite the critical importance of viruses in shaping marine microbial ecosystems and lubricating upper ocean biogeochemical cycles, relatively little is known about the molecular mechanisms mediating phytoplankton host-virus interactions. Recent work in algal host-virus systems has begun to shed novel insight into the elegant strategies of viral infection and subcellular regulation of cell fate, which not only reveal tantalizing aspects of viral replication and host resistance strategies but also provide new diagnostic tools toward elucidating the impact of virus-mediated processes in the ocean. Widespread lateral gene transfer between viruses and their hosts plays a prominent role in host-virus diversification and in the regulation of host-virus infection mechanisms by allowing viruses to manipulate and 'rewire' host metabolic pathways to facilitate infection.

Resource dynamics during infection of Micromonas pusilla by virus MpV-Sp1.

Viruses infecting marine phytoplankton drive phytoplankton diversity, terminate blooms and shuttle genetic material. Assessments of the scale of viral impacts on trophic networks are, however, speculative. We investigated fluxes of DNA between host and virus during infection of the prasinophyte alga Micromonas pusilla by phycodnavirus MpV SP1. Under a light-dark regimen, viral genomes accumulated to a transient peak within 24 h, at the expense of both host DNA synthesis and nuclear DNA. Viral genome abundance then declined soon after host lysis. This release of a phosphate-rich nucleotide pool during viral infection of phytoplankton should be considered in trophic models. Lysis required light and was stalled in darkness, meanwhile viral genome replication proceeded slowly in the dark. Viral exploitation of this host is therefore only partially light-dependent and infected phytoplankton are poised to lyse at dawn or if mixed to the photic zone. The chloroplast genome remained intact until lysis, indicating that either this DNA pool is inaccessible or the virus spares the chloroplast for its energy and reductant generation. The photochemical turnover of residual Photosystem II complexes accelerated during lysis, indicating that events in late infection heighten demands on the remaining host photosynthetic systems, consistent with the light dependency of lysis.