Viral Assemblages Research Articles

Separation of single-stranded DNA, double-stranded DNA and RNA from an environmental viral community using hydroxyapatite chromatography.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community's genetic signature. Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s. By exploiting the charge interaction between the positively-charged Ca(2+) ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromatography.

In situ structuring of virioplankton through ­bacterial exoenzymatic activity: interaction with phytoplankton

The abundance and composition of microbial communities were investigated during 2007 in Marennes Oleron Bay (France) in order to characterize the biological relationships governing the planktonic food web. Compared to the results of previous years, there was a lower abundance and a lower magnitude of variations in autotrophic and heterotrophic microbial entities in 2007. Using a Spearman rank correlation analysis, a significant link for biological abundance was only noted between bacteria and ciliates. However, an interesting relationship was highlighted between chlorophyll a and bacterial aminopeptidase and β-glucosidase exoenzymatic activities. Two charac- teristic periods of virus-bacteria uncoupling were observed in spring and autumn 2007. Using multi- variate analysis, the clustering of monthly viral community structures (defined by RAPD-fingerprint- ing) was related to bacterial proteolysis activity and secondarily to flagellates. Spring and autumn were characterized by phytoplanktonic blooms of large (>10 µm) and small cells (<10 µm), respec- tively, during the virus-bacteria uncoupling phases. High predation by flagellates in the autumn, during a period of microbial food web activity, involved the highest viral fingerprint richness, infer- ring a top-down control in driving the number of virus-host systems. We discuss the proteolysis level as a descriptor in structuring viral assemblages and argue that changes in the quantity and quality of DOM may bridge phytoplankton to bacteria and reflect changes in phytoplankton composition.

Milne Fiord epishelf lake: A coastal Arctic ecosystem vulnerable to climate change

Milne Fiord in the Canadian High Arctic contains the last known ice-dammed fiord lake (epishelf lake) in the Northern Hemisphere. This freshwater ecosystem is retained by the Milne Ice Shelf and is underlain by sea water that is connected to the Arctic Ocean. Using microscopy, photosynthetic pigment analyses, and molecular techniques we examined the planktonic communities present in Milne Fiord to determine the biotic characteristics of the epishelf lake and the sea water below. Net sampling of the water column of Milne Fiord revealed a mixture of marine, freshwater, and brackish zooplankton taxa, and high performance liquid chromatography (HPLC) pigment analysis showed pronounced differences in phytoplankton composition through the highly stratified water column. Chlorophytes dominated in the epishelf lake, prasinophytes prevailed in the halocline, and the bottom layer harboured mainly fucoxanthin-containing groups. Clone libraries of a dark-incubated, concentrated sample from below the halocline (30 m depth) yielded marine Archaea (mainly Crenarchaeota) and known bacterial taxa from the Pacific and Arctic oceans (e.g., Roseobacter, Oleispira, Colwellia). An equivalent sample from the epishelf lake (5 m depth) yielded many bacterial taxa that are characteristic of cold, freshwater habitats (e.g., Polynucleobacter, Variovorax, Flavobacterium), the euryhaline genus Polaromonas, and freshwater eukaryotes, notably ciliates. Similarly, denaturing gradient gel electrophoresis (DGGE) analyses of T4-like bacteriophages showed different viral assemblages in the upper and lower water column. This diverse, stratified ecosystem is dependent on the integrity of the bounding ice shelf and is therefore vulnerable to the ongoing effects of climate change in this region.

Using CRISPRs as a metagenomic tool to identify microbial hosts of a diffuse flow hydrothermal vent viral assemblage

Metagenomic analyses of viruses have revealed widespread diversity in the viriosphere, but it remains a challenge to identify specific hosts for a viral assemblage. To address this problem, we analyze the viral metagenome of a northeast Pacific hydrothermal vent with a comprehensive database of spacers derived from the clustered regularly interspaced short palindromic repeat (CRISPR) putative immune system. CRISPR spacer matches to the marine vent virome suggest that viruses infecting hosts from diverse taxonomic groups are present in this vent environment. Comparative virome analyses show that CRISPR spacers from vent isolates and from thermophiles in general have a higher percentage of matches to the vent virome than to other marine or terrestrial hot spring viromes. However, a high percentage of hits to spacers from mesophilic hosts, combined with a moderately high modeled alpha diversity, suggest that the marine vent virome is comprised of viruses that have the potential to infect diverse taxonomic groups of multiple thermal regimes in both the bacterial and the archaeal domains.

Physical fractionation of aquatic viral assemblages

Aquatic viruses are extraordinarily diverse and have major influences on plankton ecology and evolution, but the majority of these viruses remain uncharacterized. Most viruses have not been cultivated and cultivationindependent means to explore viral diversity have provided only a fragmented view of viral genomic information. In this study, ultracentrifugation and chromatographic methods were evaluated for their ability to fractionate aquatic viruses based on their differing physical properties with the aim of physically enriching subsets of aquatic viruses for further study. Centrifugation in continuous cesium chloride gradients, strong and weak anion‐exchange chromatography, and size‐exclusion chromatography with Sephacryl S‐1000 were found to separate aquatic viruses based on their differing buoyant densities, surface charges, and sizes, respectively. Sucrose density gradients, cation‐exchange chromatography, and size‐exclusion chromatography using controlled‐pore glass were found to have insufficient resolution to effectively separate viral assemblages. Using the successful methods listed above, we were able to separate complex assemblages of viruses into fractions that had distinguishable subsets of the viruses present in the original community as determined by pulsed‐field gel electrophoresis. Strong anion‐exchange chromatography was particularly effective at separating viruses in the samples and resulted in one to six dominant viral genome bands in most fractions. Our results suggest that individual populations of viruses may be physically enriched, and possibly isolated, from complex assemblages without cultivation. These fractionation methods are expected to improve our ability to characterize the genotypes and phenotypes of the uncultivated majority of viruses in aquatic environments.

Viral Distribution and Life Strategies in the Bach Dang Estuary, Vietnam

Although the structure and dynamics of planktonic viruses in freshwater and seawater environments are relatively well documented, little is known about the occurrence and activity of these viruses in estuaries, especially in the tropics. Viral abundance, life strategies, and morphotype distribution were examined in the Bach Dang Estuary (Vietnam) during the dry season in 2009. The abundance of both viruses and their prokaryotic hosts decreased significantly from upstream to downstream, probably as the result of nutrient dilution and osmotic stress faced by the freshwater communities. The antibiotic mitomycin-C revealed that the fraction of lysogenic cells was substantially higher in the lower seawater part of the estuary (max 27.1%) than in the upper freshwater area where no inducible lysogens were observed. The question of whether there is a massive, continuous induction of marine lysogens caused by the mixing with freshwater is considered. Conversely, the production of lytic viruses declined as salinity increased, indicating a spatial succession of viral life strategies in this tropical estuary. Icosahedral tailless viruses with capsids smaller than 60 nm dominated the viral assemblage throughout the estuary (63.0% to 72.1% of the total viral counts), and their distribution was positively correlated with that of viral lytic production. Interestingly, the gamma-proteobacteria explained a significant portion of the variance in the <60 nm and 60 to 90 nm tailless viruses (92% and 80%, respectively), and in the Myoviridae (73%). Also, 60% of the variance of the tailless larger viruses (>90 nm) was explained by the beta-proteobacteria. Overall, these results support the view that the environment, through selection mechanisms, probably shapes the structure of the prokaryotic community. This might be in turn a source of selection for the virioplankton community via specific affiliation favoring particular morphotypes and life strategies.

Viral abundance and activity in the deep sub-seafloor biosphere

Subsurface abundance and distribution of viruses and prokaryotes was determined along a depth profile, down to 96 m below seafloor (96 mbsf), at Challenger Mound from the Porcubine Seabight (IODP Expedition 307). Viral and prokary- otic abundance decreased exponentially with sedi- ment depth from 1.0 × 10 8 viruses cm -3 and 3.8 × 10 6 cells cm -3 at 4 mbsf to 4.9 × 10 6 viruses cm -3 and 9.8 × 10 5 cells cm -3 at 96 mbsf. The age of the sediment ranges from ca. 0.5 million yr before present (Ma) at 4 mbsf to ca. 2 Ma at 96 mbsf. Assuming that the decline in viral abundance with depth reflects a gradual decay of the viral assemblage over time, the estimated decay rate of the viral community is 1.2 × 10 -6 ± 0.3 × 10 -6 (SD) yr -1 , corresponding to a half-life of the viral community of 5.8 × 10 5 yr. Measurements of viral and prokaryotic change in abundance were performed in incubations of undi- luted, but homogenized, sediment samples (13.3 and 79.8 mbsf) in anaerobic bags. Viral abundance de- creased rapidly (decay rates of 0.010 ± 0.002 (SD) and 0.022 ± 0.018 (SD) h -1 , respectively) in the incu- bations, suggesting that homogenization exposed the viruses to degradation processes. We hypothe- size that most of the deep subsurface viral communi- ties inhabit a microenvironment where the viruses are protected against decay, and can therefore per- sist in undisturbed sediments for hundreds of thou- sands, perhaps even millions, of years.

Is the Genetic Landscape of the Deep Subsurface Biosphere Affected by Viruses?

Viruses are powerful manipulators of microbial diversity, biogeochemistry, and evolution in the marine environment. Viruses can directly influence the genetic capabilities and the fitness of their hosts through the use of fitness factors and through horizontal gene transfer. However, the impact of viruses on microbial ecology and evolution is often overlooked in studies of the deep subsurface biosphere. Subsurface habitats connected to hydrothermal vent systems are characterized by constant fluid flux, dynamic environmental variability, and high microbial diversity. In such conditions, high adaptability would be an evolutionary asset, and the potential for frequent host–virus interactions would be high, increasing the likelihood that cellular hosts could acquire novel functions. Here, we review evidence supporting this hypothesis, including data indicating that microbial communities in subsurface hydrothermal fluids are exposed to a high rate of viral infection, as well as viral metagenomic data suggesting that the vent viral assemblage is particularly enriched in genes that facilitate horizontal gene transfer and host adaptability. Therefore, viruses are likely to play a crucial role in facilitating adaptability to the extreme conditions of these regions of the deep subsurface biosphere. We also discuss how these results might apply to other regions of the deep subsurface, where the nature of virus–host interactions would be altered, but possibly no less important, compared to more energetic hydrothermal systems.

Metagenomic Analysis of the Viral Communities in Fermented Foods

Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.

The metavirome of a hypersaline environment

Hypersaline environments harbour the highest number of virus-like particles reported for planktonic systems. However, very little is known about the genomic diversity of these virus assemblages since most of the knowledge on halophages is based on the analysis of a few isolates infecting strains of hyperhalophilic Archaea that may not be representatives of the natural microbiota. Here, we report the characterization, through a metagenomic approach, of the viral assemblage inhabiting a crystallizer pond (CR30) from a multi-pond solar saltern in Santa Pola (SE Spain). A total of 1.35 Mbp were cloned that yielded a total of 620 kb sequenced viral DNA. The metavirome was highly diverse and different from virus communities of marine and other aquatic environments although it showed some similarities with metaviromes from high-salt ponds in solar salterns in San Diego (SW USA), indicating some common traits between high-salt viromes. A high degree of diversity was found in the halophages as revealed by the presence of 2479 polymorphic nucleotides. Dinucleotide frequency analysis of the CR30 metavirome showed a good correlation with GC content and enabled the establishment of different groups, and even the assignment of their putative hosts: the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber.

Changing phosphorus concentration and subsequent prophage induction alter composition of a freshwater viral assemblage

Summary1. The effects of nutrients on the temporal variation in viral assemblage composition, and in particular the occurrence of temperate phages, were assessed in mesotrophic Lake Erken over 5 months of the ice‐free period. The percentage of the bacterial community that contained inducible prophages (lysogenic bacteria, LB) changed over the season, being lowest in late spring and highest in early autumn. The most important variables for predicting LB were concentrations of total nitrogen (TN), total phosphorus (TP) and temperature.2. The viral assemblage composition, as determined by pulsed‐field gel electrophoresis (PFGE), also changed over the season. Prophages were induced by incubations with mitomycin C and we show, for the first time for natural communities, that the resulting temperate phages could be detected using PFGE.3. A substantial fraction (19%) of the number of detected operational taxonomic units (OTUs: defined as unique genome sizes) appeared unique to temperate phages and 41% of OTUs increased in relative abundance after treatment with mitomycin C.4. Different viral OTUs were induced at different times during the season. The most important environmental factor covarying with viral assemblage composition over the period of study, as determined by multivariate analysis, was concentration of TP. In re‐growth cultures with natural bacteria and lowered viral abundance (VA) (decreased virus to bacteria ratio), addition of PO4‐P induced prophages and resulted in subsequent production of temperate phages, as indicated by a decreased percentage of LB and increased VA. Incubations of natural bacterial communities with mitomycin C (field data) or PO4‐P (experiment) changed the viral assemblage composition at a similar rate as the observed monthly changes in the lake.

Unraveling the viral tapestry (from inside the capsid out)

The field of viral ecology has long endeavored to devise and adapt methodologies to peer beyond the visible and elucidate the roles of viruses in the environment. Much has been learned regarding the dynamics of viral assemblages and the significant role viruses have in biogeochemical cycles (Brussaard et al., 2008). Despite these advances, detailed understanding of biological processes behind ecosystem-scale effects of viral infection has remained largely obscured, with research relegated to a handful of available host–virus culture systems. Increasingly affordable DNA sequencing has provided a route to assess the genetic diversity of viruses in the environment. Current research seeks to apply genomic technologies to address knowledge gaps in environmental virology, but obstacles presented by the unique biology of viruses must be addressed to understand the context and significance of viral genome and metagenome sequence data. Coincident with the twentieth anniversary of the publication that launched the field (Bergh et al., 1989), viral ecologists from around the world met in 2009 for a workshop of the Scientific Committee for Oceanographic Research (SCOR) Working Group on the Role of Viruses in Marine Ecosystems (http://scor-viral-ecology.dbi.udel.edu) and at a session entitled ‘From Direct Counts to Metagenomics: Two Decades of Discovery in Aquatic Viral Ecology' at the 109th General Meeting of the American Society for Microbiology (ASM; http://www.asm.org). These meetings covered a broad range of topics relevant to environmental virology, however, the impact of metagenomics emerged as a major topic. Highlighted are important issues for viral metagenomics raised during a roundtable discussion (SCOR) and through various abstracts presented at both forums.

Hydroxyapatite-Mediated Separation of Double-Stranded DNA, Single-Stranded DNA, and RNA Genomes from Natural Viral Assemblages

Metagenomics can be used to determine the diversity of complex, often unculturable, viral communities with various nucleic acid compositions. Here, we report the use of hydroxyapatite chromatography to efficiently fractionate double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), dsRNA, and ssRNA genomes from known bacteriophages. Linker-amplified shotgun libraries were constructed to generate sequencing reads from each hydroxyapatite fraction. Greater than 90% of the reads displayed significant similarity to the expected genomes at the nucleotide level. These methods were applied to marine viruses collected from the Chesapeake Bay and the Dry Tortugas National Park. Isolated nucleic acids were fractionated using hydroxyapatite chromatography followed by linker-amplified shotgun library construction and sequencing. Taxonomic analysis demonstrated that the majority of environmental sequences, regardless of their source nucleic acid, were most similar to dsDNA viruses, reflecting the bias of viral metagenomic sequence databases.

Morphological Characterization of Viruses in the Stratified Water Column of Alkaline, Hypersaline Mono Lake

Concentrations of viruses and prokaryotes in the alkaline, moderately hypersaline, seasonally stratified Mono Lake are among the highest reported for a natural aquatic environment. We used electron microscopy to test whether viral morphological characteristics differed among the epilimnion, metalimnion, and the anoxic hypolimnion of the lake and to determine how the properties of viruses in Mono Lake compare to other aquatic environments. Viral capsid size distributions were more similar in the metalimnion and hypolimnion of Mono Lake, while viral tail lengths were more similar in the epilimnion and metalimnion. The percentage of tailed viruses decreased with depth and the relative percentages of tailed phage families changed with depth. The presence of large (>125nm capsid), untailed viruses in the metalimnion and hypolimnion suggests that eukaryotic viruses are produced in these suboxic and anoxic, hypersaline environments. Capsid diameters of viruses were larger on average in Mono Lake compared to other aquatic environments, and no lemon-shaped or filamentous viruses were found, in contrast to other high-salinity or high-altitude lakes and seas. Our data suggest that the physically and chemically distinct layers of Mono Lake harbor different viral assemblages, and that these assemblages are distinct from other aquatic environments that have been studied. Furthermore, we found that filtration of a sample through a 0.22-µm pore-size filter significantly altered the distribution of viral capsid diameters and tail lengths, resulting in a relative depletion of viruses having larger capsids and longer tails. This observation highlights the potential for bias in molecular surveys of viral diversity, which typically rely on filtration through 0.2- or 0.22-µm pore-size membrane filters to remove bacteria during sample preparation.

Diel and daily fluctuations in virioplankton production in coastal ecosystems

Viruses saturate the world around us, yet a basic understanding of how viral impacts on microbial host organisms vary over days to hours, which typify the replication cycles of aquatic viruses, remains elusive. Thus, diel patterns of viral production (VP) in Chesapeake Bay surface waters were examined on five sampling dates. Day-to-day variations in VP in the Chesapeake and coastal California surface waters were also investigated. Significant variations in VP were detected over 24 h cycles during four of five studies, but rates did not vary significantly over the course of a few days in either location. Diel patterns of VP displayed seasonality with shorter viral assemblage turnover times and shorter times to maximum viral abundance in summer, implying shorter replication cycles for virus-host systems in warmer months. No correlation was found between VP and time of day, likely due to seasonal changes in the diel patterns of VP. This analysis significantly increases our knowledge of the short-term patterning of in situ VP, and thus viral impacts, and suggests that variations in viral biology in response to changes in host communities or physio-chemical properties affect both diel and seasonal cycles and magnitudes of VP.

Viruses: a vast reservoir of genetic diversity and driver of global processes

Viruses are by far the most abundant ''lifeforms'' in the ocean; they exist wherever life is found. The estimated 1030 viruses in the ocean, if stretched end-to-end, would span farther than the nearest 60 galaxies. Every second there are about 1023 viral infections in the ocean. These infections are a major source of mortality; they influence the composition of marine communities, cause disease in organisms ranging from shrimp to whales, and drive biogeochemical cycles [1,2]. Each infection potentially introduces new genetic information into an organism, or into progeny viruses, and thereby drives the evolution of the host and viral assemblages. Our understanding of the impact of viruses on global systems continues to unfold, overthrowing the paradigm that viruses and viral-mediated processes are sidebars to global processes. My presentation will explore the current state of knowledge on the abundance and diversity of viruses in the sea, and the role that viruses play on nutrient and energy cycles. Gene-based and metagenomic approaches have revealed enormous genetic richness in marine viral assemblages. For example, sequences of highly-conserved genes from environmental samples have shown vast genetic richness within related groups of viruses, and exposed the existence of many genetically distinct groups for which there are no cultured representatives.

Seasonal Dynamics and Metagenomic Characterization of Estuarine Viriobenthos Assemblages by Randomly Amplified Polymorphic DNA PCR

Direct enumeration and genetic analyses indicate that aquatic sediments harbor abundant and diverse viral communities. Thus far, synecological analysis of estuarine sediment viral diversity over an annual cycle has not been reported. This oversight is due in large part to a lack of molecular genetic approaches for assessing viral diversity within a large collection of environmental samples. Here, randomly amplified polymorphic DNA PCR (RAPD-PCR) was used to examine viral genotypic diversity within Chesapeake Bay sediments. Using a single 10-mer oligonucleotide primer for all samples, RAPD-PCR analysis of sediment viral assemblages yielded unique banding patterns across spatial and temporal scales, with the occurrence of specific bands varying among the sample set. Cluster analysis of RAPD-PCR amplicon banding patterns indicated that sediment viral assemblages changed with season and to a lesser extent with geographic location. Sequence analysis of RAPD-PCR amplicons revealed that 76% of sediment viral sequences were not homologous to any sequence in the GenBank nonredundant protein database. Of the GenBank sequence homologs, the majority belonged to viruses within the Podoviridae (24%) and Myoviridae (22%) viral families, which agrees with the previously observed frequencies of these morphological families in Chesapeake Bay sediments. Furthermore, the majority of the sediment viral sequences homologous to GenBank nonredundant protein sequences were phages or prophages (57%). Hence, RAPD-PCR proved to be a reliable and useful approach for characterization of viral assemblages and the genetic diversity of viruses within aquatic sediments.

Viral communities associated with healthy and bleaching corals

The coral holobiont is the integrated assemblage of the coral animal, its symbiotic algae, protists, fungi and a diverse consortium of Bacteria and Archaea. Corals are a model system for the study of symbiosis, the breakdown of which can result in disease and mortality. Little is known, however, about viruses that infect corals and their symbionts. Here we present metagenomic analyses of the viral communities associated with healthy and partially bleached specimens of the Caribbean reef-building coral Diploria strigosa. Surprisingly, herpes-like sequences accounted for 4–8% of the total sequences in each metagenome; this abundance of herpes-like sequences is unprecedented in other marine viral metagenomes. Viruses similar to those that infect algae and plants were also present in the coral viral assemblage. Among the phage identified, cyanophages were abundant in both healthy and bleaching corals and vibriophages were also present. Therefore, coral-associated viruses could potentially infect all components of the holobiont – coral, algal and microbial. Thus, we expect viruses to figure prominently in the preservation and breakdown of coral health.

Phylogenetic Analysis of Members of the Phycodnaviridae Virus Family, Using Amplified Fragments of the Major Capsid Protein Gene

Algal viruses are considered ecologically important by affecting host population dynamics and nutrient flow in aquatic food webs. Members of the family Phycodnaviridae are also interesting due to their extraordinary genome size. Few algal viruses in the Phycodnaviridae family have been sequenced, and those that have been have few genes in common and low gene homology. It has hence been difficult to design general PCR primers that allow further studies of their ecology and diversity. In this study, we screened the nine type I core genes of the nucleocytoplasmic large DNA viruses for sequences suitable for designing a general set of primers. Sequence comparison between members of the Phycodnaviridae family, including three partly sequenced viruses infecting the prymnesiophyte Pyramimonas orientalis and the haptophytes Phaeocystis pouchetii and Chrysochromulina ericina (Pyramimonas orientalis virus 01B [PoV-01B], Phaeocystis pouchetii virus 01 [PpV-01], and Chrysochromulina ericina virus 01B [CeV-01B], respectively), revealed eight conserved regions in the major capsid protein (MCP). Two of these regions also showed conservation at the nucleotide level, and this allowed us to design degenerate PCR primers. The primers produced 347- to 518-bp amplicons when applied to lysates from algal viruses kept in culture and from natural viral communities. The aim of this work was to use the MCP as a proxy to infer phylogenetic relationships and genetic diversity among members of the Phycodnaviridae family and to determine the occurrence and diversity of this gene in natural viral communities. The results support the current legitimate genera in the Phycodnaviridae based on alga host species. However, while placing the mimivirus in close proximity to the type species, PBCV-1, of Phycodnaviridae along with the three new viruses assigned to the family (PoV-01B, PpV-01, and CeV-01B), the results also indicate that the coccolithoviruses and phaeoviruses are more diverged from this group. Phylogenetic analysis of amplicons from virus assemblages from Norwegian coastal waters as well as from isolated algal viruses revealed a cluster of viruses infecting members of the prymnesiophyte and prasinophyte alga divisions. Other distinct clusters were also identified, containing amplicons from this study as well as sequences retrieved from the Sargasso Sea metagenome. This shows that closely related sequences of this family are present at geographically distant locations within the marine environment.

Randomly Amplified Polymorphic DNA PCR as a Tool for Assessment of Marine Viral Richness

Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.