Genomics discovery of giant fungal viruses from subsurface oceanic crustal fluids.

The oceanic igneous crust is a vast reservoir for microbial life, dominated by diverse and active bacteria, archaea, and fungi. Archaeal and bacterial viruses were previously detected in oceanic crustal fluids at the Juan de Fuca Ridge (JdFR). Here we report the discovery of two eukaryotic Nucleocytoviricota genomes from the same crustal fluids by sorting and sequencing single virions. Both genomes have a tRNATyr gene with an intron (20 bps) at the canonical position between nucleotide 37 and 38, a common feature in eukaryotic and archaeal tRNA genes with short introns (<100 bps), and fungal genes acquired through horizontal gene transfer (HGT) events. The dominance of Ascomycota fungi as the main eukaryotes in crustal fluids and the evidence for HGT point to these fungi as the putative hosts, making these the first putative fungi-Nucleocytoviricota specific association. Our study suggests active host-viral dynamics for the only eukaryotic group found in the subsurface oceanic crust and raises important questions about the impact of viral infection on the productivity and biogeochemical cycling in this ecosystem.

Single-virus genomics and beyond.

Viruses are extremely diverse and modulate important biological and ecological processes globally. However, much of viral diversity remains uncultured and yet to be discovered. Several powerful culture-independent tools, in particular metagenomics, have substantially advanced virus discovery. Among those tools is single-virus genomics, which yields sequenced reference genomes from individual sorted virus particles without the need for cultivation. This new method complements virus culturing and metagenomic approaches and its advantages include targeted investigation of specific virus groups and investigation of genomic microdiversity within viral populations. In this Review, we provide a brief history of single-virus genomics, outline how this emergent method has facilitated advances in virus ecology and discuss its current limitations and future potential. Finally, we address how this method may synergistically intersect with other single-virus and single-cell approaches.

Tear Down the Fluorescent Curtain: A New Fluorescence Suppression Method for Raman Microspectroscopic Analyses.

The near exponential proliferation of published Raman microspectroscopic applications over the last decade bears witness to the strengths and versatility of this technology. However, laser-induced fluorescence often severely impedes its application to biological samples. Here we report a new approach for near complete elimination of laser-induced background fluorescence in highly pigmented biological specimens (e.g., microalgae) enabling interrogation by Raman microspectroscopy. Our simple chemiphotobleaching method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination. Our approach demonstrates the potential for confocal Raman microspectroscopy becoming an indispensable tool to obtain spatially-resolved data on the chemical composition of highly fluorescent biological samples from individual cells to environmental samples.

Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses.

Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270-14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.

Physiological responses of Oxyrrhis marina to a diet of virally infected Emiliania huxleyi.

The coccolithophore Emiliania huxleyi forms some of the largest phytoplankton blooms in the ocean. The rapid demise of these blooms has been linked to viral infections. E. huxleyi abundance, distribution, and nutritional status make them an important food source for the heterotrophic protists which are classified as microzooplankton in marine food webs. In this study we investigated the fate of E. huxleyi (CCMP 374) infected with virus strain EhV-86 in a simple predator-prey interaction. The ingestion rates of Oxyrrhis marina were significantly lower (between 26.9 and 50.4%) when fed virus-infected E. huxleyi cells compared to non-infected cells. Despite the lower ingestion rates, O. marina showed significantly higher growth rates (between 30 and 91.3%) when fed infected E. huxleyi cells, suggesting higher nutritional value and/or greater assimilation of infected E. huxleyi cells. No significant differences were found in O. marina cell volumes or fatty acids profiles. These results show that virally infected E. huxleyi support higher growth rates of single celled heterotrophs and in addition to the "viral shunt" hypothesis, viral infections may also divert more carbon to mesozooplankton grazers.

Parasite Microbiome Project: Systematic Investigation of Microbiome Dynamics within and across Parasite-Host Interactions.

Understanding how microbiomes affect host resistance, parasite virulence, and parasite-associated diseases requires a collaborative effort between parasitologists, microbial ecologists, virologists, and immunologists. We hereby propose the Parasite Microbiome Project to bring together researchers with complementary expertise and to study the role of microbes in host-parasite interactions. Data from the Parasite Microbiome Project will help identify the mechanisms driving microbiome variation in parasites and infected hosts and how that variation is associated with the ecology and evolution of parasites and their disease outcomes. This is a call to arms to prevent fragmented research endeavors, encourage best practices in experimental approaches, and allow reliable comparative analyses across model systems. It is also an invitation to foundations and national funding agencies to propel the field of parasitology into the microbiome/metagenomic era.

Genomic exploration of individual giant ocean viruses.

Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.

An Agar-Based Method for Plating Marine Protozoan Parasites of the Genus Perkinsus.

The genus Perkinsus includes protozoan parasites of mollusks responsible for losses in the aquaculture industry and hampering the recovery of natural shellfish beds worldwide, and they are a key taxon for understanding intracellular parasitism adaptations. The ability to propagate the parasite in liquid media, in the absence of the host, has been crucial for improving understanding of its biology; however, alternative techniques to grow the parasite are needed to explore other basic aspects of the Perkinsus spp. biology. We optimized a DME: Ham's F12-5% FBS- containing solid agar medium for plating Perkinsus marinus. This solid medium supported trophozoite propagation both by binary fission and schizogony. Colonies were visible to the naked eye 17 days after plating. We tested the suitability of this method for several applications, including the following: 1) Subcloning P. marinus isolates: single discrete P. marinus colonies were obtained from DME: Ham's F12-5% FBS- 0.75% agar plates, which could be further propagated in liquid medium; 2) Subcloning engineered Perkinsus mediterraneus MOE[MOE]: GFP by streaking cultures on plates; 3) Chemical susceptibility: Infusing the DME: Ham's F12-5% FBS- 0.75% agar plates with triclosan resulted in inhibition of the parasite propagation in a dose-dependent manner. Altogether, our plating method has the potential for becoming a key tool for investigating diverse aspects of Perkinsus spp. biology, developing new molecular tools, and for biotechnological applications.

Dip in the gene pool: metagenomic survey of natural coccolithovirus communities.

Despite the global oceanic distribution and recognised biogeochemical impact of coccolithoviruses (EhV), their diversity remains poorly understood. Here we employed a metagenomic approach to study the occurrence and progression of natural EhV community genomic variability. Analysis of EhV metagenomes from the early and late stages of an induced bloom led to three main discoveries. First, we observed resilient and specific genomic signatures in the EhV community associated with the Norwegian coast, which reinforce the existence of limitations to the capacity of dispersal and genomic exchange among EhV populations. Second, we identified a hyper-variable region (approximately 21kbp long) in the coccolithovirus genome. Third, we observed a clear trend for EhV relative amino-acid diversity to reduce from early to late stages of the bloom. This study validated two new methodological combinations, and proved very useful in the discovery of new genomic features associated with coccolithovirus natural communities.

Marine viruses, a genetic reservoir revealed by targeted viromics.

Metagenomics has opened new windows on investigating viral diversity and functions. Viromic studies typically require large sample volumes and filtration through 0.2 µm pore-size filters, consequently excluding or under-sampling tailed and very large viruses. We have optimized a targeted viromic approach that employs fluorescence-activated sorting and whole genome amplification to produce dsDNA-enriched libraries from discrete viral populations from a 1-ml water sample. Using this approach on an environmental sample from the Patagonian Shelf, we produced three distinct libraries. One of the virus libraries was dominated (79.65% of sequences with known viral homology) by giant viruses from the Mimiviridae and Phycodnaviridae families, while the two other viromes were dominated by smaller phycodnaviruses, cyanophages and other bacteriophages. The estimated genotypic richness and diversity in our sorted viromes, with 52-163 estimated genotypes, was much lower than in previous virome reports. Fragment recruitment of metagenome reads to selected reference viral genomes yields high genome coverage, suggesting little amplification and sequencing bias against some genomic regions. These results underscore the value of our approach as an effective way to target and investigate specific virus groups. In particular, it will help reveal the diversity and abundance of giant viruses in marine ecosystems.

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Use of microarrays to assess viral diversity: from genotype to phenotype.

The diversity among the coccolithovirus strains held in the Plymouth Virus Collection (PVC) was assessed using three complementary techniques: phylogeny based on DNA polymerase and major capsid protein gene sequence; host range; and a new, microarray-based genome-wide approach. The PVC is composed of three groups of strains that are geographically and temporally distinct. Virus strains clustered according to these groups in all three diversity assessments. Furthermore, the microarray approach based on genomic content showed that two strains, previously considered as identical to others in the PVC, are actually distinct. These results show the importance of genome-wide surveys for assessing strain diversity. Not only has the microarray provided an alternative to the phylogeny-derived pattern for virus evolution, it has also begun to provide some clues to the genes that may be responsible for the different phenotypes displayed by these viruses.

Spatial distribution and transcriptional activity of an uncultured clade of planktonic diazotrophic gamma-proteobacteria in the Arabian sea.

The spatial distribution of an uncultured clade of marine diazotrophic gamma-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these gamma-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.