Isolation of Prasinoviruses of the Green Unicellular Algae Ostreococcus spp. on a Worldwide Geographical Scale

Ostreococcus spp. are extremely small unicellular eukaryotic green algae found worldwide in marine environments, and they are susceptible to attacks by a diverse group of large DNA viruses. Several biologically distinct species of Ostreococcus are known and differ in the ecological niches that they occupy: while O. tauri (representing clade C strains) is found in marine lagoons and coastal seas, strains belonging to clade A, exemplified by O. lucimarinus, are present in different oceans. We used laboratory cultures of clonal isolates of these two species to assay for the presence of viruses in seawater samples from diverse locations. In keeping with the distributions of their host strains, we found a decline in the abundance of O. tauri viruses from a lagoon in southwest France relative to the Mediterranean Sea, whereas in the ocean, no O. tauri viruses were detected. In contrast, viruses infecting O. lucimarinus were detected from distantly separated oceans. DNA sequencing, phylogenetic analyses using a conserved viral marker gene, and a Mantel test revealed no relationship between geographic and phylogenetic distances in viruses infecting O. lucimarinus.Viruses are the most abundant and genetically diverse biological entities in marine environments (48). The three ways most often used to assess eukaryotic algal virus diversity are (i) using a functional host-virus system to quantify viruses specific to one host strain (i.e., culture-based studies) (4), (ii) using PCR amplification and sequencing a conserved gene (10, 12-14, 28), and (iii) using whole-community genome sequencing (i.e., metagenomics) (6, 8, 31). Recently, the advent of sequencing techniques like shotgun sequencing or pyrosequencing (38) has led to an increase in the number of metagenomics projects. The Global Ocean Sampling (GOS) Expedition has provided a unique opportunity to investigate viral diversity in different environments within the size fraction of 0.1 to 0.8 μm (39). The GOS data revealed highly abundant viral sequences (at least 3% of the predicted proteins had a viral origin) (53). In another study, the analysis of marine viromes from four oceanic regions suggested that the composition of viral assemblages depends on their geographic locations, but these authors conclude that similar viruses are widespread throughout the oceans (2). Despite these new methods and different ways to analyze viral diversity, we still do not really know if “everything is really everywhere” (7).The present study addresses a specific part of this problem: are viruses infecting a single host strain present at geographically distant locations? If several viral strains are identified and characterized, how closely do these viruses resemble one another on a phylogenetic scale? In order to answer these questions, we focused on a microalgal (Prasinophyceae)-virus (Prasinovirus) system. The studied hosts belong to the genus Ostreococcus, a ubiquitous prasinophyte picoeukaryotic alga abundant throughout the oceanic euphotic zone (55, 56). Several strains from this genus were isolated and assigned to four distinct ecotypes according to their growth parameters under different light regimens (22, 36), which correspond to four well-defined phylogenetic clades in an internal transcribed spacer (ITS)-based phylogeny (clades A to D). The complete genome sequences of two Ostreococcus species have been described: O. tauri (19) and O. lucimarinus (35). In the present study, viruses infecting specific host species (Ostreococcus spp.) have been screened from a variety of locations around the world.Among viruses infecting Ostreococcus, the genome of a single strain (OtV5) has been fully sequenced (18), and the phylogenetic relationships among several virus strains have been investigated (4). These viruses belong to the Prasinovirus group, a genus of the Phycodnaviridae family. Many viruses infecting phytoplankton are members of the Phycodnaviridae; they have double-stranded DNA genomes and large polyhedral capsids (20). In the prasinophyte-Prasinovirus system, the hosts and viruses can be grown on solid medium and are easily maintained in the laboratory. Ostreococcus viral strains have been isolated and characterized by phylogenetic analysis based on their B-family DNA polymerase (DNA pol) partial gene sequence (4). This DNA polymerase is a useful marker for phylogenetic analyses because its sequence is well conserved in all known members of nucleocytoplasmic large DNA viruses (NCLDVs) (26), including Phycodnaviridae. Furthermore, several previous studies have examined the abundance and the genetic diversity of marine eukaryotic viruses using environmental sequencing approaches and amplified DNA pol gene fragments (11, 12, 43-46), and Monier et al. (31) used this marker to describe the taxonomic distribution of large DNA viruses from the GOS data.The first stage of this study was to isolate Ostreococcus viruses from different worldwide geographic locations, by culturing on various host strains. In a second stage, these viral strains were characterized via the sequencing of their pol sequence (encoding a part of their DNA polymerase gene), and their specificity toward different host strains was assessed in order to assess the potential host range of the viral strains isolated and to gain a better understanding of their population dynamics and distribution. Finally we compared these new Prasinovirus DNA sequences with metagenomic sequence data (obtained from sampling all around the world) and environmental sequence data identified using BLAST similarity to assess the global distribution of similar Ostreococcus viruses.