Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus
© Ogata et al; licensee BioMed Central Ltd. 2009
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 dinoflagellate H. 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 family Phycodnaviridae 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: YS DTDS was found in HcDNAV PolB instead of YG DTDS 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 . 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 . 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 .
HcDNAV is a marine giant virus (or "girus" [6, 7]) containing dsDNA genome, and lytically infects H. circularisquama [8, 9]. HcDNAV is considered to play a significant role in the demise of H. 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 . HcDNAV is the sole DNA virus currently isolated from dinoflagellates, and to our knowledge, is the only DNA virus isolated from the superphylum Alveolata . Based on its host range, genome type/size and microscopic features, HcDNAV was previously suggested to be a member of Phycodnaviridae . However, there has been no molecular data supporting this tentative classification.
Phycodnaviridae includes intensively-studied algal virus members such as chlorella viruses and Emiliania huxleyi viruses [14–17], and belongs to a larger group of eukaryotic DNA viruses called NCLDVs . 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 are Mimiviridae, Poxviridae, Iridoviridae, and Asfarviridae. Mimiviridae is represented by the freshwater amoeba-infecting mimivirus  and its close relative mamavirus . Based on the sequences of PolB, the most conserved NCLDV core genes, three algal viruses have been suggested to belong to Mimiviridae . Poxviridae include a number of successful pathogens known to infect a tremendous variety of terrestrial animals, such as insects, reptiles, birds, and mammals . Iridoviruses infect invertebrate and cold-blooded vertebrate hosts, and includes numerous emerging pathogens of fishes and amphibians . The last family Asfarviridae [24, 25] is currently represented by a sole species, African swine fever virus (ASFV) with a 170 kbp dsDNA genome . 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 . 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-infected H. circularisquama on 6 dpi), virus particles were collected as described in . 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 , 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 of Pyramimonas 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  (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 YS DTDS- instead of the YG DTDS- 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 .
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 the Phycodnaviridae .
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 the Phycodnaviridae family . 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 , 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.
- 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. 10.1126/science.1095964View ArticlePubMedGoogle Scholar
- Gray MW, Lang BF, Burger G: Mitochondria of protists. Annu Rev Genet 2004, 38: 477-524. 10.1146/annurev.genet.37.110801.142526View ArticlePubMedGoogle Scholar
- Hallegraeff GM: A review of harmful algal blooms and their apparent global increase. Phycologia 1993, 32: 79-99.View ArticleGoogle Scholar
- 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. 10.1186/1476-069X-7-S2-S2PubMed CentralView ArticlePubMedGoogle Scholar
- Matsuyama Y: Harmful effect of dinoflagellate Heterocapsa circularisquama on shellfish aquaculture in Japan. Jpn Agr Res Quart 1999, 33: 283-293.Google Scholar
- 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. 10.1016/j.virusres.2006.01.008View ArticlePubMedGoogle Scholar
- Claverie JM, Ogata H: Ten good reasons not to exclude giruses from the evolutionary picture. Nat Rev Microbiol 2009, 7: 615. author reply 615 10.1038/nrmicro2108-c3View ArticlePubMedGoogle Scholar
- 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. 10.3354/ame023103View ArticleGoogle Scholar
- 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. 10.1128/AEM.69.5.2580-2586.2003PubMed CentralView ArticlePubMedGoogle Scholar
- 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.Google Scholar
- 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. 10.1128/AEM.71.7.3599-3607.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Nagasaki K: Dinoflagellates, diatoms, and their viruses. J Microbiol 2008, 46: 235-243. 10.1007/s12275-008-0098-yView ArticlePubMedGoogle Scholar
- Wilson WH, Van Etten JL, Schroeder DS, Nagasaki K, Brussaard C, Delaroque N, Bratbak G, Suttle CA: Phycodnaviridae. In 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.Google Scholar
- Yamada T, Onimatsu H, Van Etten JL: Chlorella viruses. Adv Virus Res 2006, 66: 293-336. 10.1016/S0065-3527(06)66006-5PubMed CentralView ArticlePubMedGoogle Scholar
- Van Etten JL: Unusual life style of giant chlorella viruses. Annu Rev Genet 2003, 37: 153-195. 10.1146/annurev.genet.37.110801.143915View ArticlePubMedGoogle Scholar
- 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. full_textPubMed CentralPubMedGoogle Scholar
- 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. 10.1101/gr.091686.109PubMed CentralView ArticlePubMedGoogle Scholar
- Iyer LM, Balaji S, Koonin EV, Aravind L: Evolutionary genomics of nucleo-cytoplasmic large DNA viruses. Virus Res 2006, 117: 156-184. 10.1016/j.virusres.2006.01.009View ArticlePubMedGoogle Scholar
- Claverie JM, Abergel C, Ogata H: Mimivirus. Curr Top Microbiol Immunol 2009, 328: 89-121. full_textPubMedGoogle Scholar
- 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. 10.1038/nature07218View ArticlePubMedGoogle Scholar
- 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. 10.1186/1743-422X-5-12PubMed CentralView ArticlePubMedGoogle Scholar
- 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. 10.1128/JVI.77.13.7590-7600.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Chinchar VG, Hyatt A, Miyazaki T, Williams T: Family Iridoviridae: poor viral relations no longer. Curr Top Microbiol Immunol 2009, 328: 123-170. full_textPubMedGoogle Scholar
- Dixon LK, Escribano JM, Martins C, Rock DL, Salas ML, Wilkinson PJ: Asfarviridae. In 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.Google Scholar
- Salas M: African swine fever virus (Asfarviridae). In Encyclopedia of Virology. 2nd edition. Edited by: Granoff A, Webster RG. London: Elsevier Academic Press; 1999:30-38.View ArticleGoogle Scholar
- 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. 10.1006/viro.1995.1149View ArticlePubMedGoogle Scholar
- 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. 10.1099/vir.0.83343-0View ArticlePubMedGoogle Scholar
- 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. 10.1016/0888-7543(95)80010-JView ArticlePubMedGoogle Scholar
- Ogata H, Raoult D, Claverie JM: A new example of viral intein in Mimivirus. Virol J 2005., 2: 10.1186/1743-422X-2-8Google Scholar
- 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. 10.1126/science.1101485View ArticlePubMedGoogle Scholar
- 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. 10.1371/journal.pbio.0050077PubMed CentralView ArticlePubMedGoogle Scholar
- Monier A, Claverie JM, Ogata H: Taxonomic distribution of large DNA viruses in the sea. Genome Biol 2008, 9: R106. 10.1186/gb-2008-9-7-r106PubMed CentralView ArticlePubMedGoogle Scholar
- 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. 10.1006/viro.1996.0234View ArticlePubMedGoogle Scholar
- 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. 10.1073/pnas.0808985105PubMed CentralView ArticlePubMedGoogle Scholar
- Van Etten JL, Lane LC, Meints RH: Viruses and viruslike particles of eukaryotic algae. Microbiol Rev 1991, 55: 586-620.PubMed CentralPubMedGoogle Scholar
- 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. 10.1016/j.tree.2005.09.005View ArticlePubMedGoogle Scholar
- Falkowski PG, de Vargas C: Genomics and evolution. Shotgun sequencing in the sea: a blast from the past? Science 2004, 304: 58-60. 10.1126/science.1097146View ArticlePubMedGoogle Scholar
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003, 52: 696-704. 10.1080/10635150390235520View ArticlePubMedGoogle Scholar
- Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, et al.: Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 2008, 36: W465-469. 10.1093/nar/gkn180PubMed CentralView ArticlePubMedGoogle Scholar
- 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. 10.1006/jmbi.2000.4042View ArticlePubMedGoogle Scholar
- 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. 10.1093/nar/25.24.4876PubMed CentralView ArticlePubMedGoogle Scholar
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