Iron Chloride Flocculation of Bacteriophages from Seawater.

Viruses influence ecosystem dynamics by modulating microbial host population dynamics, evolutionary trajectories and metabolic outputs. While they are ecologically important across diverse ecosystems, viruses are challenging to study due to minimal biomass often obtained when sampling natural communities. Here we describe a technique using chemical flocculation, filtration and resuspension to recover bacteriophages from seawater and other natural waters. The method uses iron to precipitate viruses which are recovered by filtration onto large-pore size membranes and then resuspended using a buffer containing magnesium and a reductant (ascorbic acid or oxalic acid) at slightly acid pH (6-6.5). The recovery of bacteriophages using iron flocculation is efficient (>90%), inexpensive and reliable, resulting in preparations that are amenable to downstream analysis by next generation DNA sequencing, proteomics and, in some cases, can be used to study virus-host interactions.

Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition.

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses.

BACKGROUND: Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). METHODS: Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. RESULTS: Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. DISCUSSION: Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses.

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting 'global ocean virome' dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Ocean plankton. Structure and function of the global ocean microbiome.

Microbes are dominant drivers of biogeochemical processes, yet drawing a global picture of functional diversity, microbial community structure, and their ecological determinants remains a grand challenge. We analyzed 7.2 terabases of metagenomic data from 243 Tara Oceans samples from 68 locations in epipelagic and mesopelagic waters across the globe to generate an ocean microbial reference gene catalog with >40 million nonredundant, mostly novel sequences from viruses, prokaryotes, and picoeukaryotes. Using 139 prokaryote-enriched samples, containing >35,000 species, we show vertical stratification with epipelagic community composition mostly driven by temperature rather than other environmental factors or geography. We identify ocean microbial core functionality and reveal that >73% of its abundance is shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

Ocean plankton. Patterns and ecological drivers of ocean viral communities.

Viruses influence ecosystems by modulating microbial population size, diversity, metabolic outputs, and gene flow. Here, we use quantitative double-stranded DNA (dsDNA) viral-fraction metagenomes (viromes) and whole viral community morphological data sets from 43 Tara Oceans expedition samples to assess viral community patterns and structure in the upper ocean. Protein cluster cataloging defined pelagic upper-ocean viral community pan and core gene sets and suggested that this sequence space is well-sampled. Analyses of viral protein clusters, populations, and morphology revealed biogeographic patterns whereby viral communities were passively transported on oceanic currents and locally structured by environmental conditions that affect host community structure. Together, these investigations establish a global ocean dsDNA viromic data set with analyses supporting the seed-bank hypothesis to explain how oceanic viral communities maintain high local diversity.

Viral tagging reveals discrete populations in Synechococcus viral genome sequence space.

Microbes and their viruses drive myriad processes across ecosystems ranging from oceans and soils to bioreactors and humans. Despite this importance, microbial diversity is only now being mapped at scales relevant to nature, while the viral diversity associated with any particular host remains little researched. Here we quantify host-associated viral diversity using viral-tagged metagenomics, which links viruses to specific host cells for high-throughput screening and sequencing. In a single experiment, we screened 10(7) Pacific Ocean viruses against a single strain of Synechococcus and found that naturally occurring cyanophage genome sequence space is statistically clustered into discrete populations. These population-based, host-linked viral ecological data suggest that, for this single host and seawater sample alone, there are at least 26 double-stranded DNA viral populations with estimated relative abundances ranging from 0.06 to 18.2%. These populations include previously cultivated cyanophage and new viral types missed by decades of isolate-based studies. Nucleotide identities of homologous genes mostly varied by less than 1% within populations, even in hypervariable genome regions, and by 42-71% between populations, which provides benchmarks for viral metagenomics and genome-based viral species definitions. Together these findings showcase a new approach to viral ecology that quantitatively links objectively defined environmental viral populations, and their genomes, to their hosts.

Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics.

Viruses have global impact through mortality, nutrient cycling and horizontal gene transfer, yet their study is limited by complex methodologies with little validation. Here, we use triplicate metagenomes to compare common aquatic viral concentration and purification methods across four combinations as follows: (i) tangential flow filtration (TFF) and DNase + CsCl, (ii) FeCl3 precipitation and DNase, (iii) FeCl3 precipitation and DNase + CsCl and (iv) FeCl3 precipitation and DNase + sucrose. Taxonomic data (30% of reads) suggested that purification methods were statistically indistinguishable at any taxonomic level while concentration methods were significantly different at family and genus levels. Specifically, TFF-concentrated viral metagenomes had significantly fewer abundant viral types (Podoviridae and Phycodnaviridae) and more variability among Myoviridae than FeCl3 -precipitated viral metagenomes. More comprehensive analyses using protein clusters (66% of reads) and k-mers (100% of reads) showed 50-53% of these data were common to all four methods, and revealed trace bacterial DNA contamination in TFF-concentrated metagenomes and one of three replicates concentrated using FeCl3 and purified by DNase alone. Shared k-mer analyses also revealed that polymerases used in amplification impact the resulting metagenomes, with TaKaRa enriching for 'rare' reads relative to PfuTurbo. Together these results provide empirical data for making experimental design decisions in culture-independent viral ecology studies.

Contrasting life strategies of viruses that infect photo- and heterotrophic bacteria, as revealed by viral tagging.

Ocean viruses are ubiquitous and abundant and play important roles in global biogeochemical cycles by means of their mortality, horizontal gene transfer, and manipulation of host metabolism. However, the obstacles involved in linking viruses to their hosts in a high-throughput manner bottlenecks our ability to understand virus-host interactions in complex communities. We have developed a method called viral tagging (VT), which combines mixtures of host cells and fluorescent viruses with flow cytometry. We investigated multiple viruses which infect each of two model marine bacteria that represent the slow-growing, photoautotrophic genus Synechococcus (Cyanobacteria) and the fast-growing, heterotrophic genus Pseudoalteromonas (Gammaproteobacteria). Overall, viral tagging results for viral infection were consistent with plaque and liquid infection assays for cyanobacterial myo-, podo- and siphoviruses and some (myo- and podoviruses) but not all (four siphoviruses) heterotrophic bacterial viruses. Virus-tagged Pseudoalteromonas organisms were proportional to the added viruses under varied infection conditions (virus-bacterium ratios), while no more than 50% of the Synechococcus organisms were virus tagged even at viral abundances that exceeded (5 to 10×) that of their hosts. Further, we found that host growth phase minimally impacts the fraction of virus-tagged Synechococcus organisms while greatly affecting phage adsorption to Pseudoalteromonas. Together these findings suggest that at least two contrasting viral life strategies exist in the oceans and that they likely reflect adaptation to their host microbes. Looking forward to the point at which the virus-tagging signature is well understood (e.g., for Synechococcus), application to natural communities should begin to provide population genomic data at the proper scale for predictively modeling two of the most abundant biological entities on Earth. Viral study suffers from an inability to link viruses to hosts en masse, and yet delineating "who infects whom" is fundamental to viral ecology and predictive modeling. This article describes viral tagging-a high-throughput method to investigate virus-host interactions by combining the fluorescent labeling of viruses for "tagging" host cells that can be analyzed and sorted using flow cytometry. Two cultivated hosts (the cyanobacterium Synechococcus and the gammaproteobacterium Pseudoalteromonas) and their viruses (podo-, myo-, and siphoviruses) were investigated to validate the method. These lab-based experiments indicate that for most virus-host pairings, VT (viral tagging) adsorption is equivalent to traditional infection by liquid and plaque assays, with the exceptions being confined to promiscuous adsorption by Pseudoalteromonas siphoviruses. These experiments also reveal variability in life strategies across these oceanic virus-host systems with respect to infection conditions and host growth status, which highlights the need for further model system characterization to break open this virus-host interaction "black box."

Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method.

Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (< 1 ng) starting genomic material for sequencing. Current solutions for amplifying sufficient DNA for metagenomics analyses include linear amplification for deep sequencing (LADS), which requires more DNA than is normally available, linker-amplified shotgun libraries (LASLs), which is prohibitively low throughput, and whole-genome amplification, which is significantly biased and thus non-quantitative. Here, we adapt the LASL approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a 'reconditioning PCR' step to increase yield and minimize late-cycle PCR artefacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with G + C content amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent, the first of which we tested and present data for here.

Milky hemolymph syndrome (MHS) in spiny lobsters, penaeid shrimp and crabs.

Black tiger shrimp Penaeus monodon, European shore crab Carcinus maenas and spiny lobster Panulirus spp. can be affected by milky hemolymph syndrome (MHS). Four rickettsia-like bacteria (RLB) isolates of MHS originating from 5 geographical areas have been identified to date. The histopathology of the disease was characterized and a multiplex PCR assay was developed for detection of the 4 bacterial isolates. The 16S rRNA gene and 16-23S rRNA intergenic spacer region (ISR) were used to examine the phylogeny of the MHS isolates. Although the pathology of this disease appears similar in the various different hosts, sequencing and examination of the phylogenetic relationships reveal 4 distinct RLB involved in the infection process.

Infectious myonecrosis virus has a totivirus-like, 120-subunit capsid, but with fiber complexes at the fivefold axes.

Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-A resolution. Results reveal a totivirus-like, 120-subunit T = 1 capsid, 450 A in diameter, but with fiber complexes protruding a further 80 A at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.

Purification and characterization of infectious myonecrosis virus of penaeid shrimp.

The causative agent of myonecrosis affecting cultured Penaeus vannamei in Brazil was demonstrated to be a virus after purification of the agent from infected shrimp tissues. Purified viral particles were injected into specific pathogen-free P. vannamei, resulting in a disease that displayed the same characteristics as those found in the original shrimp used for purification. The virus was named infectious myonecrosis virus (IMNV). The viral particles were icosahedral in shape and 40 nm in diameter, with a buoyant density of 1.366 g ml(-1) in caesium chloride. The genome consisted of a single, double-stranded (dsRNA) molecule of 7560 bp. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 5' ORF (ORF 1, nt 136-4953) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsRNA-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kDa. The 3' ORF (ORF 2, nt 5241-7451) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsRNA virus family that infects invertebrate hosts.

Detection of infectious myonecrosis virus (IMNV) of penaeid shrimp by reverse-transcriptase polymerase chain reaction (RT-PCR).

Infectious myonecrosis virus (IMNV) infecting cultured Litopenaeus vannamei in Brazil is a double-stranded RNA virus that causes a slowly progressive disease with cumulative mortalities of up to 70%. The disease is currently diagnosed using a combination of gross signs (primarily skeletal tail muscle necrosis with white opaque discoloration), histopathology, and in situ hybridization with a digoxigenin-labeled gene probe. A rapid and sensitive method for definitive diagnosis of the disease was developed using reverse-transcriptase polymerase chain reaction (RT-PCR). Two primer sets were used to detect 328 and 139 bp amplicons in a nested RT-PCR assay. Using RNA extracted from purified virions, the first step reaction detected 100 copies of the IMNV viral genome whereas the nested step detected 10 copies. The primers were shown to be specific for IMNV and no amplicons were detected using RNA extracted from shrimp infected with other penaeid shrimp viruses (Taura syndrome virus [TSV], yellowhead virus [YHV], infectious hypodermal hematopoietic necrosis virus [IHHNV] and white spot syndrome virus [WSSV]).

Taura syndrome virus from Belize represents a unique variant.

A Taura syndrome virus (TSV) isolate from cultured Penaeus vannamei grown in Belize, Central America was characterized and shown to be a unique isolate. Mortality rates in laboratory infections of specific pathogen-free (SPF) P. vannamei, reactivity of the virus with monoclonal antibody (MAb) 1A1 and phylogenetic analysis demonstrated that the Belize isolate (BLZ02TSV) is a new valiant of TSV. The Hawaiian 1994 TSV isolate (HI94TSV, GenBank AF277675) was used as the reference isolate for these studies. Laboratory infections of SPF P. vannamei with BLZ02TSV demonstrated higher mortalities and earlier onset of mortalities compared to infections with HI94TSV. Shrimp tissues infected with BLZ02TSV reacted with a TSV-specific gene probe by in situ hybridization and were positive by RT-PCR using TSV diagnostic primers, thus indicating that the isolate was TSV. However, Western blot analysis and immunohistochemistry using MAb 1A1 demonstrated that BLZ02TSV did not react with the antibody, suggestive of changes in the VP1 region of the genome that codes for the polypeptide to which MAb 1A1 binds. Phylogenetic analysis of a 1.3 kbp fragment of the TSV VP1 capsid region revealed that BLZ02TSV represents a distinct group among more than 29 isolates of TSV studied thus far. This research demonstrates that BLZ02TSV is a unique isolate of TSV and reiterates a problem related to the use of MAb 1A1 for detection of TSV in clinical specimens.

In situ hybridization demonstrates that Litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to experimental infection with infectious myonecrosis virus (IMNV).

Infectious myonecrosis virus (IMNV) was recently found to be the cause of necrosis in the skeletal muscle of farm-reared Litopenaeus vannamei from northeastern Brazil. Nucleic acid extracted from semi-purified IMN virions showed that this virus contains a 7.5 kb RNA genome. A cDNA library was constructed, and a clone, designated as IMNV-317, was labeled with digoxigenin-11-dUTP and used as a gene probe for in situ hybridization (ISH). This probe specifically detected IMNV in infected tissues. To determine the susceptibility of 3 species of penaeid shrimp (L. vannamei, L. stylirostris, Penaeus monodon) to IMNV infection, juveniles were injected with purified virions and observed for clinical signs of infection and mortality over a 4 wk period. All L. vannamei exhibited typical lesions after 6 d, and lesions were visible in all L. stylirostris by Day 13. The clinical signs of opaque muscle were not seen in P. monodon, due to their highly pigmented exoskeleton precluding visual detection of lesions. Moderate mortality (20%) occurred in infected L. vannamei. No mortalities were observed in either L. stylirostris or P. monodon. Histological examination and ISH indicated that all 3 species are susceptible to IMNV infection. Using ISH, IMNV was detected in tissues including the skeletal muscle, lymphoid organ, hindgut, and phagocytic cells within the hepatopancreas and heart. In all 3 species, skeletal muscle cells produced the strongest ISH reactions. Based on the onset of clinical signs of infection and mortality, L. vannamei appears to be the most susceptible of these 3 species to IMNV infection.

Geographic variations among infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolates and characteristics of their infection.

Nucleotide sequence variations of a 2.9 kb fragment of infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolated from samples of Penaeus monodon were determined and compared with an isolate from Hawaii. The infection characteristics of these isolates were examined by histology, in situ hybridization, and laboratory challenge studies with P. vannamei. Isolates of IHHNV were obtained from samples collected from the SE Asia region (the Philippines, Thailand, and Taiwan). Isolates of putative IHHNV were obtained from African samples (Tanzania, Madagascar, and Mauritius). The Philippine isolate had a very high nucleotide sequence identity (99.8%) to Hawaii IHHNV. The Thailand isolate showed a slightly lower identity (96.2%). The putative IHHNV sequences collected from Tanzania and Madagascar showed greater divergence from Hawaii IHHNV, 8.2% difference for Tanzania and 14.1% difference for Madagascar. A phylogenetic analysis showed that the Philippine IHHNV clustered with IHHNV found in the western hemisphere. This supports the theory that the Philippines was the origin of IHHNV that was first detected in Hawaii. In the laboratory infection study, both the Philippine and Thailand IHHNV were passed into P. vannamei, and the infected shrimp did not suffer any mortalities. In another laboratory infection, P. vannamei injected with a tissue homogenate of P. monodon from Madagascar, which tested positive for IHHNV by PCR, did not demonstrate IHHNV infection, suggesting that this putative IHHNV is not infectious to P. vannamei.

Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses.

The single-stranded genomic RNA of Taura syndrome virus (TSV) is 10205 nucleotides in length, excluding the 3' poly(A) tail, and contains two large open reading frames (ORFs) that are separated by an intergenic region of 207 nucleotides. The ORFs are flanked by a 377 nucleotide 5' untranslated region (UTR) and a 226 nucleotide 3' UTR followed by a poly(A) tail. The predicted amino acid sequence of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of several plant and animal RNA viruses. In addition, a short amino acid sequence located in the N-terminal region of ORF1 presented a significant similarity with a baculovirus IAP repeat (BIR) domain of inhibitor of apoptosis proteins from double-stranded DNA viruses and from animals. The presence of this BIR-like sequence is the first reported in a single-stranded RNA virus, but its function is unknown. The N-terminal amino acid sequence of three TSV capsid proteins (55, 40 and 24 kDa) were mapped in ORF2, which is not in the same reading frame as ORF1 and possesses an AUG codon upstream of the structural genes. However, the intergenic region shows nucleotide sequence similarity with those of the genus Cricket paralysis-like viruses, suggesting a similar non-AUG-mediated translation mechanism. The structure of the TSV genome [5' UTR-non-structural proteins-intergenic UTR-structural proteins-3' UTR-poly(A) tail] is similar to those of small insect-infecting RNA viruses, which were recently regrouped into a new virus genus, Cricket paralysis-like viruses.