Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus

  • Hiroyuki Ogata1,

    Affiliated with

    • Kensuke Toyoda2,

      Affiliated with

      • Yuji Tomaru2,

        Affiliated with

        • Natsuko Nakayama2,

          Affiliated with

          • Yoko Shirai2,

            Affiliated with

            • Jean-Michel Claverie1 and

              Affiliated with

              • Keizo Nagasaki2Email author

                Affiliated with

                Virology Journal20096:178

                DOI: 10.1186/1743-422X-6-178

                Received: 24 September 2009

                Accepted: 27 October 2009

                Published: 27 October 2009


                Heterocapsa circularisquama DNA virus (HcDNAV; previously designated as HcV) is a giant virus (girus) with a ~356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellateH. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the familyPhycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments (Phycodnaviridae,Mimiviridae), one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YSDTDS was found in HcDNAV PolB instead of YGDTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments.


                Dinoflagellates (Dinophyceae) are one of the highly abundant and ubiquitous unicellular eukaryotic ("protistan") components in marine environments [1]. They constitute a major class of eukaryotes within the Alveolata, a firmly established deep phylogenetic lineage that includes other diverse classes of protists, such as apicomplexans and ciliates [2]. Some dinoflagellates are autotrophic using photosynthesis, some are heterotrophic using endocytotic feeding, and many dinoflagellates are mixotrophic having both modes of nutrition. Blooms of certain photosynthetic dinoflagellates kill fish and bivalves, or pollute shellfishes for food with particular toxins, and can lead to serious economic damages in aquaculture [3, 4].Heterocapsa circularisquama forms blooms causing massive death of shellfish such as pearl oysters and mussels, and is one of the most intensively studied dinoflagellate species [5].

                HcDNAV is a marine giant virus (or "girus" [6, 7]) containing dsDNA genome, and lytically infectsH. circularisquama [8, 9]. HcDNAV is considered to play a significant role in the demise ofH. circularisquama blooms [9, 10]. HcDNAV has a large icosahedral capsid (180-210 nm in diameter), which packs a ~356-kbp genome [8, 11]. During its multiplication, virions emerge from a specific cytoplasm compartment, called "viroplasm", which is created by the virus [9]. HcDNAV is the sole DNA virus currently isolated from dinoflagellates, and to our knowledge, is the only DNA virus isolated from the superphylum Alveolata [12]. Based on its host range, genome type/size and microscopic features, HcDNAV was previously suggested to be a member ofPhycodnaviridae [13]. However, there has been no molecular data supporting this tentative classification.

                Phycodnaviridae includes intensively-studied algal virus members such as chlorella viruses andEmiliania huxleyi viruses [1417], and belongs to a larger group of eukaryotic DNA viruses called NCLDVs [18]. NCLDVs complete their replication cycle within the host cytoplasm, and share an array of conserved core genes for transcription, RNA processing, replication, DNA packaging, and structural components. Other viral families of NCLDVs areMimiviridae,Poxviridae,Iridoviridae, andAsfarviridae.Mimiviridae is represented by the freshwater amoeba-infecting mimivirus [19] and its close relative mamavirus [20]. Based on the sequences of PolB, the most conserved NCLDV core genes, three algal viruses have been suggested to belong toMimiviridae [21].Poxviridae include a number of successful pathogens known to infect a tremendous variety of terrestrial animals, such as insects, reptiles, birds, and mammals [22]. Iridoviruses infect invertebrate and cold-blooded vertebrate hosts, and includes numerous emerging pathogens of fishes and amphibians [23]. The last familyAsfarviridae [24, 25] is currently represented by a sole species, African swine fever virus (ASFV) with a 170 kbp dsDNA genome [26]. ASFV is a large (~200 nm in diameter), intracytoplasmically-replicating arbovirus, naturally maintained in a sylvatic cycle between wild swine (warthogs and bushpigs) and argasid ticks (Ornithodoros). In these hosts, ASFV infection is usually asymptomatic [27]. However, ASFV causes an acute hemorrhagic infection in domestic swine with mortality rates up to 100% for some viral isolates.

                In an attempt to further characterize HcDNAV, we performed a low coverage shotgun sequencing of its genome. Specifically, from 4 liters of HcDNAV suspension (lysate of HcDNAV-infectedH. circularisquama on 6 dpi), virus particles were collected as described in [11]. The viral genomic DNA was purified in a PFGE-gel and was subjected to shotgun sequencing (coverage = 0.11 X). Resulting sequence reads covered part of the region containing a PolB-like sequence. With the use of tail-PCR method [28], we successfully determined a 5,800 bp sequence (DDBJ accession number AB522601) containing an open reading frame (ORF) for the complete HcDNAV PolB gene. By means of a reverse transcription-PCR (RT-PCR) experiment, the PolB gene was shown to be transcribed to mRNA (additional file 1); thus, it is most likely crucial for the replication of HcDNAV.

                HcDNAV PolB gene was found to be 3,675 bp long (forward strand, position = nt 1,913-5,590 in AB522601), punctuated by normal start and stop codons, and no intron or intein-like sequence was observed. The predicted protein product is 1,225 amino acids (aa) long. Unexpectedly, the translated amino acid sequence showed the closest BLASTP hits against PolB sequences from different ASFV isolates, with the best homolog being DPOL_ASFL6 (identity = 27%, bit score = 311, E-value = 4.10E-82) in the NCBI non-redundant sequence database. The best non-ASFV hit corresponded to the PolB sequence ofPyramimonas orientalis virus (DPOL_POV01, identity = 23%, bit score = 131, E-value = 4.10E-28). A multiple sequence alignment of the HcDNAV PolB and its close homologs confirmed the presence of conserved residues for exonuclease and polymerase activities [29] (additional file 2). Curiously, the HcDNAV PolB sequence exhibited a rarely observed amino acid substitution within the motif containing two highly conserved metal binding aspartic acid residues; HcDNAV exhibits the motif YSDTDS- instead of the YGDTDS- sequence usually found in dsDNA viruses. In addition, we identified two ORFs in the upstream region of the PolB ORF in a divergent orientation. Their products were respectively predicted to be 245 and 194 aa in length (positions = nt 463-1,200 and 1,255-1,839). The former showed a significant similarity to HNH endonucleases with its BLASTP best hit to mimivirus L245 (YP_142599, E-value = 4E-11); the latter showed a significant similarity to hypothetical proteins from NCLDVs with its best hit to mimivirus R325 (annotated as a metal-dependent hydrolase, YP_142679.1, E-value = 1E-12). Incidentally, R325 is located near the PolB gene (R322) in the mimivirus genome [30].

                To examine the unexpected sequence similarity between the HcDNAV and ASFV PolBs, we conducted a series of maximum likelihood phylogenetic analyses. First, we aligned the HcDNAV PolB sequence with its homologs from NCLDVs. A phylogenetic tree based on the 362 amino acid residue sites from the alignment supported the monophyletic grouping of HcDNAV and ASFV with a 100% bootstrap value (Fig. 1). The grouping of each of the other four NCLDV families was also supported by a high bootstrap value (100% forIridoviridae, 81% forPhycodnaviridae, 90% forMimiviridae and 100% forPoxviridae). Next, we used a wider range of viral homologs including those of bacteriophages. The resulting tree based on 320 amino acid residues again supported the grouping of HcDNAV/ASFV with a 98% bootstrap value (Fig. 2).
                Figure 1

                Maximum likelihood tree of PolB amino acid sequences from NCLDVs. Alignment was constructed with the use of T-Coffee. All the gap-containing amino acid residue sites were removed before tree construction. The phylogenetic tree was constructed using PhyML [38] available at [39] using WAG matrix and gamma distribution. Branch labels indicate bootstrap percentages (≥ 50%) after 100 replicates. The tree is essentially an unrooted tree, albeit mid-point rooted only for presentation purpose. The same method was used for the phylogenetic trees in Fig. 2, Fig. 3 and in the additional file 3. HcDNAV and ASFV sequences are indicated by filled diamond marks. CeV: Chrysochromulina ericina virus; PoV: Pyramimonas orientalis virus; HaV: Heterosigma akashiwo virus.

                Figure 2

                Maximum likelihood tree of PolB amino acid sequences from diverse groups of viruses. HcDNAV and ASFV sequences are indicated by filled diamond marks.

                In addition, we obtained a short sequence partially corresponding to an RNA polymerase II large subunit gene from HcDNAV genomic DNA (AB522602), for which we obtained a similar result. The 892 bp sequence showed BLASTX best hit against ASFV RNA polymerase sequence (RPB1_ASFM2, E-value = 2E-12). A monophyletic grouping between the HcDNAV sequence (97 aa) and the ASFV RNA polymerase sequence was again received a high bootstrap value of 87% (additional file 3).

                Our homology search and phylogenetic analyses thus confirm that the newly determined HcDNAV sequences are most closely related to their ASFV homologs. This result is in clear contradiction with the previous proposal that HcDNAV may belong to thePhycodnaviridae [13].

                A previous "phylogenetic mapping" survey of the metagenomic sequence data sets generated by the Global Ocean Sampling (GOS) expedition [31] revealed several PolB-like sequences most closely related to the PolB sequence of ASFV [32]. This observation suggested the presence of ASFV-related viruses in marine environments. In order to examine whether the "ASFV-like" marine PolB sequences were close to the HcDNAV PolB sequence, we retrieved 267 sequences from the environmental sequence collection of NCBI/GenBank using the PolB sequences of HcDNAV and ASFV as queries (E-value < 1E-10). These environmental sequences were in turn searched against the NCBI non-redundant sequence database and the HcDNAV PolB sequence. Of the 267 sequences, 15 showed their best hit to the ASFV PolB, one showed its best hit to HcDNAV (gi|136563424), and the remaining sequences had their best hit to other viruses or cellular organisms. Therefore, most of the ASFV/HcDNAV-like PolB sequences in the marine environmental collection are more closely related to the ASFV PolB than to the HcDNAV homolog. A phylogenetic tree using several environmental sequences supported their grouping with the terrestrial ASFV PolB (bootstrap value = 84%, Fig. 3).
                Figure 3

                Maximum likelihood tree of PolB amino acid sequences from NCLDVs and several sequences from environmental samples (indicated by open diamond marks). HcDNAV and ASFV sequences are indicated by filled diamond marks.

                PolB is one of the most reliable phylogenetic markers for large eukaryotic DNA viruses [32, 33]. The fact that the HcDNAV PolB was not grouped with the PolBs from phycodnaviruses strongly argues against the previous tentative classification of HcDNAV in thePhycodnaviridae family [13]. It is clear that the definitive classification of HcDNAV will require the complete sequencing of its genome. It may also turn out that the HcDNAV genome corresponds to a mosaic of NCLDV genes with different evolutionary histories, precluding a simple classification scheme. Pending its complete genome sequencing, we recently proposed to the ICTV to create a new genus "Dinodnavirus" where to tentatively classify the HcDNAV.

                Our finding now establishes an evolutionary link between a terrestrial pathogen and a marine girus. A recent metagenomic analysis of corals provided evidence for the existence of viruses related to herpesviruses [34], which have been mostly isolated as pathogens of terrestrial animals. So far, giruses of 7 algal classes [12, 35] have been isolated; still, we know next to nothing about viruses infecting other protists in aquatic environments. Given the huge diversity of protists [36, 37], a comparable diversity probably exists for marine viruses living in these environments. Exploring this hidden viral world is necessary to our understanding of the evolutionary relationships between aquatic viruses and their terrestrial relatives.



                This work was in part supported by the PACA-BioInfo Platform and Marseille-Nice Genopole.

                Authors’ Affiliations

                Information Génomique et Structurale, CNRS-UPR2589, Institut de Microbiologie de la Méditerranée, Parc Scientifique de Luminy, Aix-Marseille Université
                Harmful Algal Bloom Division, National Research Institute of Inland Sea, Fisheries Research Agency


                1. Falkowski PG, Katz ME, Knoll AH, Quigg A, Raven JA, Schofield O, Taylor FJ: The evolution of modern eukaryotic phytoplankton. Science 2004,305:354–360.View ArticlePubMed
                2. Gray MW, Lang BF, Burger G: Mitochondria of protists. Annu Rev Genet 2004,38:477–524.View ArticlePubMed
                3. Hallegraeff GM: A review of harmful algal blooms and their apparent global increase. Phycologia 1993,32:79–99.View Article
                4. Erdner D, Dyble J, Parsons ML, Stevens RC, Hubbard KA, Wrabel ML, Moore SK, Lefebvre KA, Anderson DM, Bienfang P,et al.: Centers for Oceans and Human Health: a unified approach to the challenge of harmful algal blooms. Environ Health 2008,7(Suppl 2):S2.View ArticlePubMed
                5. Matsuyama Y: Harmful effect of dinoflagellate Heterocapsa circularisquama on shellfish aquaculture in Japan. Jpn Agr Res Quart 1999,33:283–293.
                6. Claverie JM, Ogata H, Audic S, Abergel C, Suhre K, Fournier PE: Mimivirus and the emerging concept of "giant" virus. Virus Res 2006,117:133–144.View ArticlePubMed
                7. Claverie JM, Ogata H: Ten good reasons not to exclude giruses from the evolutionary picture. Nat Rev Microbiol 2009,7:615. author reply 615View ArticlePubMed
                8. Tarutani K, Nagasaki K, Itakura S, Yamaguchi M: Isolation of a virus infecting the novel shellfish-killing dinoflagellate Heterocapsa circularisquama . Aquat Microb Ecol 2001,23:103–111.View Article
                9. Nagasaki K, Tomaru Y, Tarutani K, Katanozaka N, Yamanaka S, Tanabe H, Yamaguchi M: Growth characteristics and intraspecies host specificity of a large virus infecting the dinoflagellate Heterocapsa circularisquama . Appl Environ Microbiol 2003,69:2580–2586.View ArticlePubMed
                10. Tomaru Y, Nagasaki K: Widespread occurrence of viruses lytic to the bivalve-killing dinoflagellate Heterocapsa circularisquama along the western coast of Japan. Plankton Biol Ecol 2004,51:1–6.
                11. Nagasaki K, Shirai Y, Tomaru Y, Nishida K, Pietrokovski S: Algal viruses with distinct intraspecies host specificities include identical intein elements. Appl Environ Microbiol 2005,71:3599–3607.View ArticlePubMed
                12. Nagasaki K: Dinoflagellates, diatoms, and their viruses. J Microbiol 2008,46:235–243.View ArticlePubMed
                13. Wilson WH, Van Etten JL, Schroeder DS, Nagasaki K, Brussaard C, Delaroque N, Bratbak G, Suttle CA: Phycodnaviridae. Virus Taxonomy, Eighth Report of the ICTV (Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA). London: Elsevier Academic Press 2005, 163–175.
                14. Yamada T, Onimatsu H, Van Etten JL: Chlorella viruses. Adv Virus Res 2006,66:293–336.View ArticlePubMed
                15. Van Etten JL: Unusual life style of giant chlorella viruses. Annu Rev Genet 2003,37:153–195.View ArticlePubMed
                16. Wilson WH, Van Etten JL, Allen MJ: The Phycodnaviridae: the story of how tiny giants rule the world. Curr Top Microbiol Immunol 2009,328:1–42.View ArticlePubMed
                17. Monier A, Pagarete A, de Vargas C, Allen MJ, Read B, Claverie JM, Ogata H: Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus. Genome Res 2009,19:1441–1449.View ArticlePubMed
                18. Iyer LM, Balaji S, Koonin EV, Aravind L: Evolutionary genomics of nucleo-cytoplasmic large DNA viruses. Virus Res 2006,117:156–184.View ArticlePubMed
                19. Claverie JM, Abergel C, Ogata H: Mimivirus. Curr Top Microbiol Immunol 2009,328:89–121.View ArticlePubMed
                20. La Scola B, Desnues C, Pagnier I, Robert C, Barrassi L, Fournous G, Merchat M, Suzan-Monti M, Forterre P, Koonin E, Raoult D: The virophage as a unique parasite of the giant mimivirus. Nature 2008,455:100–104.View ArticlePubMed
                21. Monier A, Larsen JB, Sandaa RA, Bratbak G, Claverie JM, Ogata H: Marine mimivirus relatives are probably large algal viruses. Virol J 2008,5:12.View ArticlePubMed
                22. Upton C, Slack S, Hunter AL, Ehlers A, Roper RL: Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome. J Virol 2003,77:7590–7600.View ArticlePubMed
                23. Chinchar VG, Hyatt A, Miyazaki T, Williams T: Family Iridoviridae: poor viral relations no longer. Curr Top Microbiol Immunol 2009,328:123–170.View ArticlePubMed
                24. Dixon LK, Escribano JM, Martins C, Rock DL, Salas ML, Wilkinson PJ: Asfarviridae. Virus Taxonomy, Eighth Report of the ICTV (Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA). London: Elsevier Academic Press 2005, 135–143.
                25. Salas M: African swine fever virus (Asfarviridae). Encyclopedia of Virology 2 Edition (Edited by: Granoff A, Webster RG). London: Elsevier Academic Press 1999, 30–38.
                26. Yáñez RJ, Rodriguez JM, Nogal ML, Yuste L, Enriquez C, Rodriguez JF, Vinuela E: Analysis of the complete nucleotide sequence of African swine fever virus. Virology 1995,208:249–278.View ArticlePubMed
                27. Chapman DAG, Tcherepanov V, Upton C, Dixon LK: Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates. J Gen Virol 2008,89:397–408.View ArticlePubMed
                28. Liu YG, Whittier RF: Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 1995,25:674–681.View ArticlePubMed
                29. Ogata H, Raoult D, Claverie JM: A new example of viral intein in Mimivirus. Virol J 2005.,2:
                30. Raoult D, Audic S, Robert C, Abergel C, Renesto P, Ogata H, La Scola B, Suzan M, Claverie JM: The 1.2-megabase genome sequence of Mimivirus. Science 2004,306:1344–1350.View ArticlePubMed
                31. Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, Wu D, Eisen JA, Hoffman JM, Remington K,et al.: The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS Biol 2007,5:e77.View ArticlePubMed
                32. Monier A, Claverie JM, Ogata H: Taxonomic distribution of large DNA viruses in the sea. Genome Biol 2008,9:R106.View ArticlePubMed
                33. Chen F, Suttle CA: Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes. Virology 1996,219:170–178.View ArticlePubMed
                34. Vega Thurber RL, Barott KL, Hall D, Liu H, Rodriguez-Mueller B, Desnues C, Edwards RA, Haynes M, Angly FE, Wegley L, Rohwer FL: Metagenomic analysis indicates that stressors induce production of herpes-like viruses in the coral Porites compressa . Proc Natl Acad Sci USA 2008,105:18413–18418.View ArticlePubMed
                35. Van Etten JL, Lane LC, Meints RH: Viruses and viruslike particles of eukaryotic algae. Microbiol Rev 1991,55:586–620.PubMed
                36. Keeling PJ, Burger G, Durnford DG, Lang BF, Lee RW, Pearlman RE, Roger AJ, Gray MW: The tree of eukaryotes. Trends Ecol Evol 2005,20:670–676.View ArticlePubMed
                37. Falkowski PG, de Vargas C: Genomics and evolution. Shotgun sequencing in the sea: a blast from the past? Science 2004,304:58–60.View ArticlePubMed
                38. Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003,52:696–704.View ArticlePubMed
                39. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M,et al.: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 2008,36:W465–469.View ArticlePubMed
                40. Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 2000,302:205–217.View ArticlePubMed
                41. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997,25:4876–4882.View ArticlePubMed

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