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Applied and Environmental Microbiology, May 2008, p. 3048-3057, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.02548-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

J. B. Larsen,* A. Larsen, G. Bratbak, and R.-A. Sandaa

Department of Biology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway

Received 12 November 2007/ Accepted 13 March 2008


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ABSTRACT
 
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.


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INTRODUCTION
 
The Phycodnaviridae family consists of large double-stranded-DNA (dsDNA) viruses infecting eukaryotic algae (5, 13). Members of the family are some of the largest known viruses, with genome lengths ranging from 170 to 560 kbp and particle sizes ranging from 100 to 220 nm (13, 37, 48, 49). Besides their extraordinary size, members and prospective members of the Phycodnaviridae are also interesting due to their ecological importance. Viruses belonging to the family have been found to infect harmful phytoplankton species, such as Heterosigma akashiwo and Phaeocystis spp. (6, 20, 32, 53). Accumulating evidence also suggests that these viruses are active players in the formation and termination of algal blooms (30), including blooms of the coccolithophorid Emiliania huxleyi (3, 28, 38).

The organization of the Phycodnaviridae viruses has previously been based on host range rather than phylogenetic analysis of virus isolates (5). However, in cases where such data are available, they largely support this clustering (31, 53). The currently valid genera in the Phycodnaviridae family are Chlorovirus, which includes the type species PBCV-1, Coccolithovirus, Phaeovirus, Prasinovirus, Prymnesiovirus, and Raphidovirus (13). The phylogenetic relationships between these genera have been difficult to establish due to a lack of genetic data and the small number of characterized viruses in the family, which is less than three for each genus except for the chloroviruses. Ecological and diversity studies of Phycodnaviridae are further complicated by the lack of a ubiquitously distributed and conserved genetic marker comparable to the rRNA genes of prokaryotic and eukaryotic microorganisms. The nucleocytoplasmic large DNA viruses (NCLDV), which include the Phycodnaviridae, have been found to share only nine common genes (19, 48, 52). Degenerate PCR primers targeting conserved motifs within the class I core genes have been used to amplify sequences from uncharacterized isolates and seawater samples (8-10). Traditionally, the most applied genetic marker for studying members of the Phycodnaviridae family has been the DNA polymerase I gene of the B family (DNA polB) (8, 10, 18). The design of PCR primers targeting the conserved region of this gene was based on the few available sequences at the time (8-10). Although successful amplification has been done from some cultured phycodnaviruses, these primers have proven unsuccessful against others (37).

Viruses of the Phycodnaviridae and Iridoviridae families are morphologically indistinguishable by electron microscopy, and this similarity is corroborated by comparison of their major capsid proteins (MCPs) (47). Analysis of the amino acid sequence of the MCP of iridoviruses has revealed seven conserved domains within this protein that are also found in at least some members of the Phycodnaviridae and in African swine fever virus (40, 47). These results implies that the MCP may be a useful genetic marker for phylogenetic inference of iridovirus ancestry (47, 50, 51). The presence of conserved interspaced domains flanked by heterologous regions suggests that the gene could also serve as a target for PCR primers and for phylogenetic analysis of the Phycodnaviridae family.

Using the mcp gene as a proxy, the aim of this work was to infer the phylogenetic relationships and genetic diversity among algal viruses in culture and marine viral assemblages. PCR primers targeting conserved regions in the mcp gene were designed, and the amplicons obtained from viruses in culture and natural seawater samples were sequenced. Phylogenetic analysis of these sequences as well as of homologous sequences retrieved from the Sargasso Sea metagenome library showed that closely related sequences are widely distributed in the marine environment. The phylogenetic relationships among the large dsDNA algal viruses inferred from the mcp and DNA polB genes were largely in agreement, but the analysis also suggested that the ancestries of these genes may be different and that the current phylogeny of Phycodnaviridae needs revision.


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MATERIALS AND METHODS
 
Primer design.
As part of an ongoing genome sequencing project, we scanned the assembled contigs of four previously isolated dsDNA viruses, Phaeocystis pouchetii virus 01 (PpV-01), Chrysochromulina ericina virus 01B (CeV-01B), Pyramimonas orientalis virus 01B (PoV-01B), and Emiliania huxleyi virus 99B1 (EhV-99B1) (7, 20, 37), for the nine type I core genes of the NCLDV group (1, 36). Except for the serine/threonine protein kinase, all type I genes were found in the four genomes. BLAST searches using the translated sequences gave closest matches to members of the Phycodnaviridae family and to mimivirus. To identify conserved nucleotides for the design of degenerate primers, we aligned the protein sequences of the type I core genes from our genome sequencing project against the sequences of other members of the Phycodnaviridae available in GenBank (HaV-1, PBCV-1, EsV-1, FirrV-1, and EhV-86), using ClustalW (43). The sequences of the most conserved proteins were compared, and we identified eight conserved motifs in the MCP, with the conserved regions II and IV having high conservation at the nucleotide level (Fig. 1). A pair of degenerate PCR primers (mcp Fwd [5'-GGY GGY CAR CGY ATT GA-3'] and mcp Rev [5'-TGI ARY TGY TCR AYI AGG TA-3']) were designed to amplify a product covering the area between these two regions (II-IV), based on the sequences from the viruses HaV-1, PBCV-1, PpV-01, PoV-01B, and CeV-01B (Fig. 1). The protein and nucleotide alignment, however, showed large differences between these viruses and EhV-99B1, EhV-86, EsV-1, and FirrV-1. Therefore, it was not possible to include the sequences of the latter group in the primer design. The degenerate primers were synthesized with the nucleotide analog inosine as a neutral base at all base positions in the primers containing four-degree degeneracy. The primers were also designed with the least possible degeneracy at the 3' end, and the quality was verified in silico using the computer program Fast PCR (Institute of Biotechnology, University of Helsinki, Finland).


Figure 1
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  		FIG. 1. Alignment of the eight conserved domains of the MCPs from 15 isolated members of the Phycodnaviridae family, together with the sequence from mimivirus. Consensus 1, consensus sequence of the viruses PBCV-1, cAR158, cNY2A, cATCV-1, cMT325, CvK-2, CvG-1, PoV-01B, PpV-01, CeV-01B, HaV-1, and mimivirus; consensus 2, consensus sequence of the total aligned sequences. The complete N-and C-terminal regions (regions I and VIII) are shown, except for the EsV-1 sequence, whose C-terminal region contains an extra 40-aa insert. The symbol "/" indicates that the sequence has been truncated. The amino acid sequences of the targets of the forward primer (region II) and reverse primer (region IV) are highlighted in the sequence of PBCV-1 (light and dark gray, respectively).

Virus lysates and concentration of seawater samples.
The ability of the primers to amplify products from different members of the Phycodnaviridae was tested on (i) cultured clonal isolates of putative Phycodnaviridae viruses, (ii) nonclonal viral lysates obtained by infecting algal cultures with concentrated seawater, and (iii) concentrated seawater samples.

PpV-01, CeV-01B, and PoV-01B were isolated in 1995 (PpV-01) (20) and 1998 (CeV-01B and PoV-01B) (37). These clonal isolates are maintained in culture in our laboratory by inoculating exponentially growing cultures of their respective algal hosts with virus lysate at an approximate ratio of 100:1. Lysates from the viruses PgV-16T, infecting Phaeocystis globosa, and MpV-12T, infecting Micromonas pusilla, were kindly provided by Joaquin Martinez-Martinez and Corina Brussard (Royal Netherlands Institute of Sea Research, The Netherlands).

Nonclonal PoV, CeV, and PpV lysates were obtained on several occasions (see Table 2) by inoculating exponentially growing cultures of Pyramimonas orientalis, Chrysochromulina ericina, and Phaeocystis pouchetii with viruses concentrated from seawater samples. These nonclonal viral lysates were kept in culture for >2 generations, with no further purification steps prior to PCR.


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  		TABLE 2. Names and sizes of sequences obtained using degenerate primers targeting the mcp genea

Algal cultures used to produce the viral lysates (clonal and nonclonal) were obtained from the culture collection at the Department of Biology, University of Bergen, and were grown in 50 ml IMR 1/2 medium (14) at 8°C (Phaeocystis pouchetii strain AJ01) or 16°C (Pyramimonas orientalis strain IFM and Chrysochromulina ericina strain IFM) with a 10-h-14-h dark-light cycle.

Seawater samples for concentration of viruses were collected from Raunefjorden (60°27'N, 5°21'E) in June 2006 and from Puddefjorden (60°33'N, 5°33'E) in August 2006. Approximately 2 liters of seawater was prefiltered through low-protein-binding 0.45-µm filters with a 142-mm diameter (Millipore, Billerica, MA). This removed zooplankton, phytoplankton, and some of the bacteria. The filtrate was then concentrated to ~45 ml, using a QuixStand benchtop system and hollow-fiber cartridges with a 100,000 pore size (NMWC; GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

PCR, cloning, and sequencing of mcp gene fragments.
PCRs were performed directly on the virus lysates and concentrated seawater samples in a total volume of 20 µl. The reaction mixture contained 1 µl virus lysate or concentrate, 10 µl HotStar master mix (Qiagen, Germany), and a 0.5 µM concentration of each of the primers. Amplification was done in a Bio-Rad Icycler machine. The program consisted of an initial hot start at 95°C for 15 min, followed by a touchdown PCR of 20 cycles of denaturation at 94°C for 30 seconds, annealing at an initial temperature of 60°C for 30 seconds, and elongation at 72°C for 30 seconds. The annealing temperature was programmed with a 0.5°C decrement per cycle (initial annealing temperature of 60°C and ending temperature of 50°C). The primary touchdown was followed by an additional 35 cycles at a constant annealing temperature of 45°C. The run was terminated after 7 min of incubation at 72°C. The PCR products were prepared for sequencing by either extraction of DNA bands from agarose gels or cloning of the reaction products. DNA was purified from agarose gels (2%) by use of a GeneClean Turbo kit (Q-BIOgene, Irvine, CA). Cloning of the PCR products was accomplished using a Strataclone PCR cloning kit (Stratagene, La Jolla, CA) following the protocol of the manufacturer. Sequencing was done at the sequencing facility at the University of Bergen. The sequences obtained have been deposited in GenBank under the reference numbers given in Tables 1 and 2.


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  		TABLE 1. Isolated members of the Phycodnaviridae family used in this study, with GenBank accession numbers for the MCP and DNA PolB protein sequences

Phylogenetic analysis.
For phylogenetic analysis, we used the protein sequences translated from the mcp genes of known Phycodnaviridae and the operational taxonomical units (OTUs) amplified from viruses in this study (Tables 1 and 2; see Fig. 3). This sequence data set was extended by alignment to the environmental database in GenBank, using the tblastN algorithm. In order to restrict the number of protein sequences used for generating phylogenetic trees, we included only the four most homologous sequences from each search. To conduct a similar search based on the DNA PolB protein, we BLAST searched the conserved region of all currently available Phycodnaviridae DNA PolB proteins. The sequence of mimivirus was included in both trees due to its high homology in the MCP to some members of the Phycodnaviridae (Fig. 1).


Figure 3
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  		FIG. 3. Phylogenetic inference using the amino acid sequence of the region of the MCP targeted by the degenerate primers from this study. The sequences of the 16 amplicons were aligned with currently available sequences of the Phycodnaviridae, the homologous sequence of mimivirus, and sequences obtained from the environmental database in GenBank. The produced tree placed the two sequences from the iridoviruses FV-3 and LCDV-1 as outgroups without additional rooting. Names in bold indicate sequences presented in this study. Genus names based on host range analysis are shown to the right of the tree. Group A, clade of virus sequences with unknown hosts; group B, clade containing viruses infecting Pyramimonas orientalis; group C, different clusters of sequences from the metagenome survey. The source of OTUs is given by the prefix M06 for amplicons from Raunefjorden, June 2006, or the prefix P06 for amplicons from samples collected from Puddefjorden, August 2006. Sequences from the Sargasso Sea metagenome are indicated by GenBank accession number alone. Nodes in the tree with bootstrap values of >75% are marked by black dots, while actual bootstrap values are shown for nodes with values between 50 and 75%. The scale bar indicate the number of amino acid substitutions per residue.

The translated amino acid sequences of the collected mcp and DNA polB gene data sets were aligned using ClustalX (43). Neighbor-joining (NJ) analysis was performed using the program PAUP 4.0 beta 10 (41), and supports for the clades were identified by means of bootstrap analysis with 1,000 replicates. Phylogenetic trees were viewed using the program Treeview, version 1.6.6 (34), and were rooted using the corresponding sequences from two iridoviruses, namely, frog virus 3 (FV-3) and lymphocystis disease virus type 1 (LCDV-1).


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RESULTS
 
By comparing the amino acid sequences of the MCPs from the phycodnaviruses PBCV-1, CvK-2, cAR158, cNY2A, cATCV-1, cMT325, CvG-1, HaV-1, PpV-01, CeV-01B and PoV-01B and from mimivirus, we identified eight conserved regions within the protein (Fig. 1). These included a large, ~66-amino-acid (aa) domain at the N terminus and a smaller, ~20-aa domain at the C-terminal end of the protein (Fig. 1, regions I and VIII). Although the identified regions largely matched conserved domains previously identified in the family Iridoviridae (40, 47), several differences, including a small region of approximately 17 aa, were observed (Fig. 1, region II). Alignment of the nucleotide sequences identified two short conserved stretches within regions II and IV as suitable targets for primer design. Using the nucleotide sequences of the mcp genes from PBCV-1, HaV-1, PoV-01B, PpV-01, and CeV-01B, we designed a forward primer with 16-degree degeneracy, annealing to region II in the gene, and a reverse primer with 512-degree degeneracy, targeting region IV. Amplification using this primer set produced fragments of 347 bp to 518 bp (Fig. 2A and B; Table 2). By PCR, we obtained OTUs from nine unknown putative Phycodnaviridae viruses amplified from seawater samples, as well as from seven isolated viruses kept in culture. However, the primers were not able to amplify a product from one Micromonas pusilla virus isolate, MpV-12T (data not shown).


Figure 2
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  		FIG. 2. PCR amplification using degenerate primers targeting part of the mcp gene. (A) Lanes 1 to 4, amplification performed on lysates from CeV-01B, PpV-01, PgV-16T, and a PoV isolate; lane 5, negative control; lane M, 100-bp marker. (B) Lane 1, amplification from concentrated viruses from a seawater sample taken at Puddefjorden, August 2006; lane M, 100-bp marker.

The unrooted NJ tree based on the fragments amplified by the degenerate primers placed the protein sequences from the iridoviruses FV-3 and LCDV-1 as outgroups (Fig. 3). The two Emiliana huxleyi viruses grouped together with the phaeoviruses EsV-1 and FirrV-1 in a cluster that branched off from the other members of the Phycodnaviridae family. This group was arranged into several distinct clusters. All of the Chlorella-infecting viruses formed a separate group in a cluster including 5 OTU sequences obtained from the current study and 13 sequences retrieved from the Sargasso Sea metagenome (Fig. 3, Chlorovirus and group A). All four sequences obtained from viruses infecting Pyramimonas orientalis also grouped together in a cluster with closest homology to sequences from the Sargasso Sea metagenome (Fig. 3, group B). A third cluster consisted of viruses infecting prymnesiophytes (putative prymnesioviruses) (Fig. 3), including two viruses infecting Phaeocystis pouchetii, one infecting P. globosa, and three infecting Chrysochromulina ericina. This group also included several sequences retrieved from the Sargasso Sea metagenome as well as one OTU sequence obtained in this study (Fig. 3, Prymnesiovirus). Several minor clusters were also observed in the tree, including 15 of the Sargasso Sea sequences and 3 sequences from the current study (Fig. 3, group C). The mimivirus and HaV-1 MCP sequences each formed its own cluster within this group (Fig. 3, Mimiviridae and Raphidovirus).

The sequences in the environmental database in GenBank that most closely matched the sequences of the Phycodnaviridae and Mimiviridae mcp genes were all from the Sargasso Sea metagenome. Several sequences from this library grouped together with the OTU sequences obtained from Raune- and Puddefjorden, generating a very diverse clade within the tree (Fig. 3, group A). Sequences closely related to the putative prymnesioviruses were also identified (Fig. 3, Prymnesiovirus). In contrast, a much higher level of diversity was observed for the mcp genes from EhV, EsV-1, FirrV-1, HaV-1, and mimivirus compared to their closest matches in GenBank.

To test the use of amplified mcp fragments as a proxy for inferring phylogenetic relationships, we generated an NJ phylogenetic tree based on the available complete protein sequences of the mcp genes from the Phycodnaviridae (Fig. 4). The tree confirmed the clustering observed from the phylogenetic analysis using the region amplified by the primers (Fig. 4).


Figure 4
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  		FIG. 4. Phylogenetic inference using the complete protein sequences of the MCPs from 16 members of the Phycodnaviridae family, together with the sequence from mimivirus. The tree was rooted using the sequences of FV-3 and LCDV-1 of the Iridoviridae family. Sequences marked in bold indicate virus sequences published in this study. The scale bar indicates the number of amino acid substitutions per residue.

To compare the phylogenetic properties of the MCP and the DNA PolB protein from phycodnaviruses, we generated an NJ phylogenetic tree by using the conserved region of the DNA PolB protein (Fig. 5). OTUs assigned to the family were obtained from GenBank by a BLAST homology search, together with the sequences from 20 viruses with known hosts (Table 1). The data set was further increased by aligning the individual sequences against the environmental database in GenBank. As was the case with the MCP sequences, all of the most homologous sequences found were from the Sargasso Sea metagenome. The phylogenetic tree was rooted using the conserved region of the DNA PolB protein from the iridoviruses FV-3 and LCDV-1. In contrast to the tree generated using MCP sequences, the EhV-86 virus and EsV-1 and FirrV-1 did not group together (Fig. 5, Coccolithovirus and Phaeovirus). The two phaeoviruses also branched off and formed a separate cluster with no close homologous sequences, while EhV-86 clustered within the tree with one only distantly related sequence from the Sargasso Sea metagenome library. PoV-01B, PpV-01, and CeV-01B formed separate clades that clustered together with mimivirus as their closest homologue with a known host (Fig. 5, groups A and B and Mimiviridae). HaV-1 was separated from the other sequences (Fig. 5, Raphidovirus). The three Phaeocystis globosa-infecting viruses and the Chrysochromulina brevifilum virus did not cluster together with PpV-01 and CeV-01B, although they all have prymnesiophyte algae as hosts (Fig. 5, Prymnesiovirus). The six sequences from viruses infecting Chlorella spp. made up one group, with no apparent close homologue identified in the environmental database in GenBank (Fig. 5, Chlorovirus). Previously identified OTUs and sequences retrieved from the Sargasso Sea metagenome made up a large cluster together with the Micromonas pusilla viruses (MpVs), which previously were assigned to their own genus (5) (Fig. 5, group C and Prasinovirus).


Figure 5
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  		FIG. 5. Phylogenetic tree based on alignment of DNA PolB protein sequences from the Phycodnaviridae family and mimivirus. Only the conserved region of the protein was used. The tree was rooted using the homologous sequences from the two iridoviruses FV-3 and LCDV-1. Previous identified OTUs are indicated by name and GenBank accession number, while sequences obtained from the Sargasso Sea metagenome are indicated by accession number alone. Sequences reported in this study are marked in bold. Nodes with >75% bootstrap values are marked by black dots, while actual bootstrap values are shown for nodes with values between 50 and 75%. Currently assigned genus names are shown to the right of the tree. The scale bar indicates the number of amino acid substitutions per residue.


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DISCUSSION
 
Using degenerate primers targeting the conserved regions II and IV of the mcp gene, we amplified nine additional mcp gene fragments from concentrated virus samples. All of these mapped phylogenetically to the Phycodnaviridae family (Fig. 3). Similar amplicons were obtained from seven isolated dsDNA viruses infecting the algae Chrysochromulina ericina, Phaeocystis pouchetii, P. globosa, and Pyramimonas orientalis.

Due to insertions/deletions between the conserved regions II, III, and IV, the amplicons produced by the primers varied in size between 347 and 518 bp (Table 2). Although it has previously been shown that some members of the Phycodnaviridae contain introns or inteins in their DNA polB genes, no such domain was identified using the CD-search algorithm (www.ncbi.nlm.nih.gov/BLAST) on the mcp gene or amplified fragments (27, 31). It therefore seems likely that the different sizes of the amplicons are due to structural differences in the protein between different viruses.

Although capable of amplifying products from most lysate and concentrated virus samples, the degenerate primers presented here are not universal for the current members of the Phycodnaviridae. Due to differences between gene sequences, it was not possible to include the nucleotide sequences of the two coccolithoviruses EhV-99B1 and EhV-86 and the two phaeoviruses EsV-1 and FirrV-1. When tested, the primers also failed to generate amplicons from one virus infecting Micromonas pusilla (MpV-12T).

The phylogenetic tree generated using the translated protein sequences of the amplified mcp gene fragments supported the existence of the currently legitimate genera within the Phycodnaviridae family. The previously unpublished sequences of CeV-01B, PpV-01, and PgV-16T formed one group and hence suggest the existence of a Prymnesiovirus genus including viruses infecting prymnesiophytes (Fig. 3, Prymnesiovirus). The four viruses infecting the prasinophyte Pyramimonas orientalis grouped into their own cluster (Fig. 3, group B). The only other dsDNA viruses which previously have been found to infect species of this algal class are viruses infecting Micromonas pusilla (8). Due to the failure of the primers to amplify products from these viruses, it was impossible to confirm the existence of the Prasinovirus genus based on the mcp sequence alone. The phylogenetic tree supported clustering of the Coccolithovirus and Phaeovirus genera, but these groups were found to diverge from the other members of the Phycodnaviridae. Similar observations based on a combined comparison of eight conserved genes (excluding the mcp gene) suggested that the coccolithoviruses should be recognized as a subfamily within the Phycodnaviridae (1). A revised phylogeny of Mimiviridae and Phycodnaviridae based on the DNA polB gene was also discussed by Monier et al. (29).

The NJ tree based on the conserved region of the DNA PolB protein sequence also maintained the current legitimate genera in the Phycodnaviridae family, but the ancestry was different from that for the MCP tree. The phaeoviruses formed the same deep branching clade as in the MCP tree, but this clade did not include the coccolithoviruses. Viruses infecting prymnesiophytes appeared in two different clusters. One included viruses assigned to the Prymnesiovirus genus (i.e., PgV-03T, PgV-06T, PgV-102P, and CbV-PW3) (2, 9, 53), and one included the prymnesiophyte-infecting viruses CeV-01B and PpV-01 as well as the prasinophyte-infecting virus PoV-01B. This is in contrast with the mcp tree, where PgV-16T clustered with the PpVs and CeVs, while the PoVs clustered outside, and could suggest possible horizontal gene transfer of DNA polB within this group. These results also corroborate earlier studies indicating a divergence of the DNA polB sequences of viruses infecting Phaeocystis globosa (53).

All of the phylogenetic analyses in this study indicate that mimivirus is more closely related to the chloroviruses, raphidoviruses, and especially the three newly assigned members CeV-01B, PpV-01, and PoV-01B than to other members of the Phycodnaviridae (Fig. 3 to 5). This differs from a previous phylogenetic analysis where mimivirus was assigned to a separate family (Mimiviridae) adjacent to the Phycodnaviridae (36). This analysis was based on the nine shared core genes of the NCLDVs, but the only Phycodnaviridae sequence data available then were from PBCV-1 and EsV-1. Based on homology in the MCP and the DNA PolB protein, mimivirus seems to be more closely related to the PoV-01B, PpV-01, CeV-01B, and PBCV-1 viruses of the Phycodnaviridae than the EhV-86, EsV-1, and FirrV-1 viruses are. A closer relationship between mimivirus and members currently assigned to the Phycodnaviridae family than was previously reported is also suggested by the fact that mimivirus contains all eight conserved domains identified in the mcp genes of the Phycodnaviridae (Fig. 1).

Many of the sequences retrieved from the Sargasso Sea metagenome showed strong homology to the sequences from the Phycodnaviridae, while others showed no relationship to any phycodnaviruses with a known host. One large cluster inferred from the MCP tree (Fig. 3, group A) branched off close to the Chlorovirus cluster. This cluster included both sequences from the Sargasso Sea metagenome and five OTUs from this study. A similar cluster branching off close to the Chlorovirus genus was also inferred from the DNA PolB sequences (Fig. 5, group C and Prasinovirus). This cluster included viruses known to infect the prasinophyte Micromonas pusilla. The presence of closely related viral sequences in as geographically distant locations as Norwegian fjords and the Sargasso Sea is in agreement with previous studies (4, 39). The relatively large number of sequences phylogenetically distinct from any known phycodnavirus may be interpreted to suggest the presence of a yet unknown genus with wide geographical distributions.

As could be expected based on their host range, no close relatives of the Chlorovirus genus were identified in the Sargasso Sea metagenome. Interestingly, this also seemed to be the case for HaV-1 and the phaeoviruses, while only a few related sequences were identified as having homology to the coccolithoviruses. This observation may reflect the small number of species identified within these families of algal hosts. For instance, only 3 Heterosigma species are currently reported in the Algaebase species list (version 4.2; http://www.algaebase.org), whereas 56 species of Chrysochromulina are listed. This could suggest a coupling of the diversity within the algal host range to the diversity in the individual genera of Phycodnaviridae viruses. However, it should be noted that the difference could also be due to geographical variations as well as to the restricted target range of the currently available universal primers targeting this family.

By applying degenerate primers targeting the mcp gene, we obtained sequences from three Chrysochromulina ericina-infecting viruses that were isolated between 1998 and 2006 and from two Phaeocystis pouchetii-infecting viruses, isolated in 1995 and 2002. The amplified sequences were 518 and 500 bp, respectively, and both sets showed 100% homology at the nucleotide level (Fig. 3, Prymnesiovirus). In contrast, the four virus isolates infecting Pyramimonas orientalis were much more divergent, with various sizes of the amplicons and sequence similarities ranging from 79 to 93% between the strains (Fig. 3, group B). Although this finding could be due to paralogous genes in PoV and none in CeV and PpV, we did not get a 100% homology hit when BLAST searching with the obtained fragments from PoVs against the available contigs of the PoV-01B genome. Thus, it seems that the evolution of at least this gene is much more constrained in the CeVs and PpVs, while the mcp gene is much more plastic in the PoVs. It is tempting to interpret this to reflect differences in virus-host interactions. Species within the algal genera Phaeocystis and, to some extent, Chrysochromulina form blooms, and the respective virus isolates are stable and easily kept in culture (unpublished observations). Pyramimonas is not a bloom former, the virus shows variable properties in culture (e.g., variable lysis and burst size), and infectivity is easily lost. Thyrhaug et al. (44, 45) showed that cultures of Phaeocystis pouchetii, Emiliana huxleyi, and Chrysochromulina ericina that recovered after viral lysis could coexist with their respective viruses at high abundances. In contrast, the concentration of PoV in recovered cultures of Pyramimonas orientalis decreased after 2 weeks (45). This could suggest that PoV particles have a higher decay rate or that P. orientalis has a lower susceptibility to viral infection. The latter theory may explain the high genetic diversity observed for PoV as a result of an increased arms race between virus and host. The alternative interpretation that the degenerate primers were able to amplify products from only one particular genotype of PpV and CeV but from many PoV genotypes seems unlikely, since the primers amplified products successfully from all the tested isolates of these viruses.

The results of this study suggest that the MCP of large dsDNA viruses can be a useful genetic marker for generating preliminary phylogenetic trees. The data also confirm the assignment of CeV-01B, PpV-01, and PoV-01B to the Phycodnaviridae family (20, 37). CeV-01B and PpV-01 infect prymnesiophytes but did not cluster with viruses in the Prymnesiovirus genus, based on the conserved region of the DNA polB gene (Fig. 5). Likewise, PoV-01B infects a prasinophyte but did not cluster with viruses infecting Micromonas pusilla, which are the only known members of the Prasinovirus genus (Fig. 5). This could imply different ancestries of the mcp and DNA polB genes of these viruses. The grouping of phycodnaviruses into genera has previously been based mainly on host range and phylogenetic analysis based on the DNA PolB protein (5, 8, 10, 22). Genome comparison has indicated that the current tree needs revision (1). Based on the phylogenetic analysis reported in this study, the status of the Coccolithovirus and Phaeovirus genera is suggested to change. Also, the results suggest that a general revision of the phylogeny of viruses belonging to the Phycodnaviridae and of mimivirus is needed. However, in order to confirm these results, a thorough comparison of the complete genomes, including phylogenetic analysis of all the core genes, is required.


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ACKNOWLEDGMENTS
 
We thank Joaquin Martinez-Martinez and Corina Brussard for supplying the lysates of the PgV-16T and MpV-12T viruses used in this study. We are also grateful to Anders Lanzen for help in the annotation of the DNA polymerase and major capsid protein genes from CeV-01B, PoV-01B, PpV-01, and EhV-99B1 and to Elinor Thompson Bartle for proofreading the manuscript.

This work was supported by grants from the Research Council of Norway to the projects "Biodiversity patterns: blooms versus stable coexistence in the lower part of marine foodwebs" (no. 158936/I10) and "Bioprospecting huge marine algal viruses."


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Biology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway. Phone: 47 55584644. Fax: 47 55589671. E-mail: jens.larsen{at}bio.uib.no Back

{triangledown} Published ahead of print on 21 March 2008. Back


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Applied and Environmental Microbiology, May 2008, p. 3048-3057, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.02548-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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