Open Access Short report

A neurotropic herpesvirus infecting the gastropod, abalone, shares ancestry with oyster herpesvirus and a herpesvirus associated with the amphioxus genome

Keith W Savin1*, Benjamin G Cocks15, Frank Wong23, Tim Sawbridge15, Noel Cogan1, David Savage14 and Simone Warner2

Author Affiliations

1 Biosciences Research Division, Department of Primary Industries, 1 Park Drive, Bundoora, Victoria 3083, Australia

2 Biosciences Research Division, Department of Primary Industries, 475 Mickleham Road, Attwood Victoria 3049, Australia

3 Australian Animal Health Laboratory, CSIRO Livestock Industries, Geelong, VIC 3220, Australia

4 School of Plant Biology, University of Western Australia, 35 Stirling Hwy Crawley, W.A 6009, Australia

5 La Trobe University, Bundoora, Victoria 3086, Australia

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Virology Journal 2010, 7:308  doi:10.1186/1743-422X-7-308

The electronic version of this article is the complete one and can be found online at: http://www.virologyj.com/content/7/1/308


Received:4 August 2010
Accepted:10 November 2010
Published:10 November 2010

© 2010 Savin et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

With the exception of the oyster herpesvirus OsHV-1, all herpesviruses characterized thus far infect only vertebrates. Some cause neurological disease in their hosts, while others replicate or become latent in neurological tissues. Recently a new herpesvirus causing ganglioneuritis in abalone, a gastropod, was discovered. Molecular analysis of new herpesviruses, such as this one and others, still to be discovered in invertebrates, will provide insight into the evolution of herpesviruses.

Results

We sequenced the genome of a neurotropic virus linked to a fatal ganglioneuritis devastating parts of a valuable wild abalone fishery in Australia. We show that the newly identified virus forms part of an ancient clade with its nearest relatives being a herpesvirus infecting bivalves (oyster) and, unexpectedly, one we identified, from published data, apparently integrated within the genome of amphioxus, an invertebrate chordate. Predicted protein sequences from the abalone virus genome have significant similarity to several herpesvirus proteins including the DNA packaging ATPase subunit of (putative) terminase and DNA polymerase. Conservation of amino acid sequences in the terminase across all herpesviruses and phylogenetic analysis using the DNA polymerase and terminase proteins demonstrate that the herpesviruses infecting the molluscs, oyster and abalone, are distantly related. The terminase and polymerase protein sequences from the putative amphioxus herpesvirus share more sequence similarity with those of the mollusc viruses than with sequences from any of the vertebrate herpesviruses analysed.

Conclusions

A family of mollusc herpesviruses, Malacoherpesviridae, that was based on a single virus infecting oyster can now be further established by including a distantly related herpesvirus infecting abalone, which, like many vertebrate viruses is neurotropic. The genome of Branchiostoma floridae (amphioxus) provides evidence for the existence of a herpesvirus associated with this invertebrate chordate. The virus which likely infected amphioxus is, by molecular phylogenetic analysis, more closely related to the other 2 invertebrate viruses than to herpesviruses infecting vertebrates (ie chordates).

Findings

In 2005 there was an outbreak of acute ganglioneuritis in an Australian population of the edible gastropod mollusc, abalone or Haliotis spp[1]. Using transmission electron microscopy, herpes-like particles were observed in ganglia of affected abalone[2] and purified virions from moribund abalone nervous tissues were identified as resembling those of herpesviruses, having an icosohedral capsid approximately 100 nm in diameter surrounded by a 150 nm diameter spiked envelope[3]. Potential herpesvirus particles were also identified previously in Taiwan following mortalities in Haliotis diversicolor [4]. Recently a diagnostic PCR test has been developed to detect the abalone virus [5]. The test has detected viral DNA sequences in diseased abalone from separate geographical locations in Australia and in DNA isolated from a herpes-like virus found some time ago in Taiwan[4].

Three herpesvirus families have been described in the order Herpesvirales - the Herpesviridae which infect Mammalia, Aves and Reptilia, the Alloherpesviridae infecting Amphibia and Osteichthyes (bony fish), and the mollusc-infecting Malacoherpesviridae containing a single virus that infects an invertebrate class, Bivalvia [6-8]. The phylogenetic relationships of these herpesviruses have been well studied and their evolution over epochs is largely synchronous with host lineages [7,8]. Gastropods separated early in the Cambrian period from all other known herpesvirus hosts. This unique evolutionary positioning[6] combined with our discovery of a related herpesvirus genome apparently integrated into the genome of another invertebrate, amphioxus, expands the Herpesvirales order and provides two key links to understanding the nature of the ancient ancestors of mollusc and vertebrate herpesviruses. To understand the structural and evolutionary relationships of the abalone virus to other herpesviruses, we purified abalone virus particles and isolated and sequenced genomic DNA using methods previously described[3,9]. The DNA was subjected to multiple displacement amplification[10] and sequenced using the Roche 454 GS-FLX system followed by partial genome assembly using the Newbler algorithm (Roche).

Based on the assembled DNA sequences of the abalone virus, several protein coding sequences predicted using Artemis[11] showed varying distant homology to herpesvirus proteins, most notably those of Ostreid herpesvirus 1 (oyster herpesvirus 1, OsHV-1), a virus infecting bivalve mollusc species[12,13]. BLAST analysis[14] of assembled sequence contigs based on predicted proteins identified 39 full length homologues of OsHV-1 genes (Table 1). These coding sequences, within partial genome scaffold sequences, or as individual coding sequences, have been submitted to Genbank. None of the coding sequences identified appear to be split by introns. Full-length sequences encoding homologues of DNA polymerase and the DNA packaging ATPase subunit of the (putative) terminase (henceforth referred to as the polymerase and terminase respectively), were identified and chosen for use in sequence alignments and phylogenetic analysis (Figures 1 & 2). Hereafter, we will refer to the new abalone virus as abalone herpesvirus or AbHV-1.

Table 1. OsHV-1 homologues of AbHV-1 coding sequences

thumbnailFigure 1. Alignment of ATP hydrolase domains from terminase protein sequences. ClustalW alignment of one of the conserved regions of the putative terminase gene - the ATP hydrolase (ATPase) domain from various herpesviruses taken from Table 3, identified using Interproscan. Grey background = >90% conserved amino acids.

thumbnailFigure 2. Dendrogram of concatenated DNA polymerase and terminase protein sequences from 34 herpesviruses. Dendrogram illustrating the evolutionary relationship of abalone and amphioxus herpesviruses to 32 other herpesviruses based on the concatenated full length protein sequences of DNA polymerase and the ATPase subunit of the putative terminase for each virus. The tree was inferred with MEGA4[32] using the Minimum Evolution (ME) method and a model based on the number of amino acid differences detected after an alignment using ClustalW[19]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The scale bar for the branch lengths = 100 amino acid sequence differences.

During the search for homologues of predicted AbHV-1 proteins using BLAST we identified, in the non-redundant (nr) Genbank protein sequence database, Branchiostoma floridae (amphioxus) coding sequences with significant homology to some of those in AbHV-1. The genome of amphioxus has been recently sequenced[15] although final assembly of chromosomes is not yet complete. On further analysis of amphioxus coding sequences using BLASTP with the predicted protein sequences of the oyster herpesvirus OsHV-1 genome (Genbank NC_005881), we identified 19 herpesvirus gene homologues. Consistent with this being an integrated virus, we found that 18 of these genes are clustered within a 150 kb region of a single amphioxus scaffold BRAFLscaffold_217, including the herpesvirus specific terminase gene[16] and all but 4 of these genes do not contain introns. These virus coding sequences appear to be legitimately assembled within published genome sequence scaffolds and are therefore probably integrated within the amphioxus genome. Further experiments such as fluorescence in situ hybridisation of chromosomes would confirm this. The 19 coding sequences identified are listed in Table 2 along with their OsHV-1 homologues and BLAST scores. We utilised the amphioxus virus terminase and polymerase protein sequence homologues in our analyses.

Table 2. Branchiostoma floridae (amphioxus) homologues of OsHV-1 coding sequences

The putative terminase, or DNA packaging ATPase, appears specific to herpesviruses and some bacteriophages, such as T4[16] and is thought to be an enzyme motor involved in packaging viral DNA into preformed capsids[17]. We used the ATPase motif from this protein to investigate the phylogeny of the herpesviruses.

The ATP hydrolase (ATPase) motif sequences from 20 of the 34 terminase proteins listed in Table 3, plus their T4 bacteriophage homologue and the amphioxus terminase homologue (XP_002591195.1, listed in Table 2), were identified using Interproscan[18] and aligned using ClustalW[19]. Figure 1 shows that 12 amino acids are conserved across all herpesvirus ATPase domain sequences, including those from the abalone, oyster and amphioxus virus genomes, indicating the placement of the abalone virus and putative amphioxus virus within the Herpesvirales order. A common ancestral origin for the mollusc and amphioxus viruses is confirmed by the absence of introns in the terminase gene and the presence of additional amino acid loops (Figure 1). Although being in the same clade (Figure 2), at a protein sequence level the mollusc viruses are only moderately related with 40% amino acid identity in this conserved viral protein, across their full length.

Table 3. Genbank Accessions of Herpesvirus Polymerase and Terminase protein sequences used for phylogenetic analysis

The phylogenetic analysis comparing concatenated polymerase and terminase full-length proteins (Figure 2, Table 3), illustrates the evolutionary relationships within the Herpesvirales order. The five Alloherpesviridae viruses are clustered together, with separate clades for frog and fish viruses as found previously [8], and the Herpesviridae are clustered into separate major clades reflecting their taxonomic groupings of alpha-, beta- and gammaherpesvirinae sub-families. The phylogenetic analysis confirms a relationship between the amphioxus virus and the abalone and oyster viruses in a deep invertebrate clade. The level of divergence makes estimation of the relative divergence times of the 3 herpesvirus families difficult. Interestingly, the amphioxus virus is in the clade with mollusc viruses, which may not have been expected given the amphioxus chordate host lineage is more aligned with vertebrates than molluscs.

The invertebrate herpesvirus clade provides a unique branching point to inform the metazoan diversification of the herpesviruses. It is thought that during the Cambrian era, the Bilaterial species diverged to generate the Protostomes (evolving into such animals as flatworms, molluscs and arthropods) and the Deuterostomes (from which the chordates and then the vertebrates evolved)[20,21]. Molluscs emerged more than 100 My before vertebrates with a bony skeleton (the current known range of herpesviruses in vertebrates). One hypothesis to explain the diversity of viruses within vertebrates and the positioning of the mollusc viruses among them, rather than as an ancestral outgroup, is the existence of diverse herpesviruses in Cambrian metazoans. Consistent with this hypothesis, previous estimates for the divergence of just the Herpesviridae in vertebrates indicate a divergence of alpha-, beta- and gammaherpesviruses to over 400 Mya, and longer times are predicted for divergence of Alloherpesviridae and Malacoherpesviridae[7]. An alternate hypothesis to explain the branching of the 3 herpesvirus families is that molluscs acquired herpesviruses by transmission in the aquatic environment, for example through association such as mollusc predation of early chordates. It appears that modern Malacoherpesviridae may have the ability to infect across species, a feature not typically observed in vertebrate herpesviruses, although the infection observed is restricted to related mollusc species[22].

As more sequence data and gene structure for Alloherpesviridae, Malacoherpesviridae and other invertebrate herpesviruses become available it will allow a more informative analysis of their evolution. Of particular interest will be new herpesviruses yet to be discovered in species which share bilateral symmetry such as amphioxus, sea squirts, flatworms or squid. Our discovery of clustered intact herpesvirus genes in amphioxus suggests an opportunistic integration has occurred in the amphioxus genome. This may not be a normal feature of infection and latency, but herpesviruses can occasionally integrate into the genome of their host[23]. Surprisingly, the nearest relatives of this chordate virus seem to be the viruses infecting molluscs rather than those of fish or frogs. Although herpesvirus particles have not been seen in the more primitive metazoan species, their existence is suspected; short herpes-like DNA sequences having been found in a metagenomic study of Hawaian coral[24]. Further metagenomic approaches similar to those described previously[25] and PCR-directed approaches[26] based on new sequences described here will enable these evolutionary questions to be addressed. The sequence information is also crucial for the development of molecular diagnostic tools to monitor and manage disease outbreaks.

The neurotropism of certain herpesviruses is well documented but this behaviour is not known outside the families of herpesviruses infecting terrestrial vertebrates[27,28]. The neurotropic tissue infection profile of the new gastropod virus analysed here is shared with some viruses within the Herpesviridae family. Convergent evolution may have given rise to the neurotropism seen in some members of the Herpesviridae and now the Malacoherpesviridae families. The rooting of a neurotropic invertebrate virus near or before the divergence of alpha-, beta-, and gammaherpesviruses, may also suggest that early mammalian herpesvirus precursors were neurotropic and that some have retained this feature over time. It is interesting to speculate as to the earliest functional interactions between sensory cells and viruses, as the first sign of neurons appeared over 600 million years ago in "cnidarians," (eg: hydra), but organisms basal to them like sponges do not have neurons or synapses[29]. Recent evidence indicates sponges have gene networks in cells which were precursors to nerve cells including proteins related to virus nerve entry receptors[30]. Others[24] have speculated on a link between herpesvirus neurotropism and the evolution of modern herpesviruses from ancestors infecting invertebrates such as Cnidaria (for example, coral or sea anemones), thought to be related to the first species with sensory receptors[31]. Further, the discovery reported here of a putative herpesvirus integrated into the genome of amphioxus hints at a wide diversity of herpesviruses within the invertebrate community, perhaps dating back to before the divergence of arthropods, molluscs and chordates. It will be exciting to discover such invertebrate herpesviruses and explore their links to ancient herpesvirus ancestors.

To accommodate the new abalone virus, which we have suggested naming abalone herpesvirus or AbHV-1, within the Herpesvirales order, we suggest the creation of a new genus called Haliotivirus within the Malacoherpesviridae family and assignment of AbHV-1 as a species under Haliotivirus (as Haliotid herpesvirus 1).

We have referred to the putative virus genome integrated into the Branchiostomid species chromosome as amphioxus-associated virus, AaHV-1. We suggest the species name Branchiostomid herpesvirus 1. Given the unique nature of the virus revealed by phylogenetic analysis and the unique evolutionary positioning of amphioxus as an invertebrate chordate, we suggest this virus, if classified, could be a member of a new family, Aspondyloherpesviridae (from the Greek for "no spine").

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

KWS, FW, BGC, SW conceived and designed the experiments; FW, NC performed the experiments; KWS, TS, DS analyzed the data; FW, SW, TS, DS, NC contributed reagents, materials, analysis tools; KWS, BGC wrote the paper. All authors have contributed to the editing or revision of the manuscript and approve its publication.

Acknowledgements

The authors wish to thank Fisheries Victoria for supplying infected abalone, German Spangenberg for facilitating the genome sequencing and Megan Vardy for technical assistance during generation of DNA sequence data. Funding was provided by the Department of Primary Industries, Victoria, Australia, The Commonwealth Scientific & Industrial Organisation, Australia and the Fisheries Research & Development Corp., Australia. The funding bodies had no role in the study design, data collection, analysis or interpretation, manuscript preparation or submission other than contributing to author salaries and experiment costs.

References

  1. Hooper C, Hardy-Smith P, Handlinger J: Ganglioneuritis causing high mortalities in farmed Australian abalone (Haliotis laevigata and Haliotis rubra).

    Australian Veterinary Journal 2007, 85:188-193. PubMed Abstract | Publisher Full Text OpenURL

  2. NACA: Quarterly Aquatic Animal Disease Report (Asia and Pacific Region) 2006/1.

    Book Quarterly Aquatic Animal Disease Report (Asia and Pacific Region) 2006/1 (Editor ed.^eds.). City 2006, 5. OpenURL

  3. Tan J, Lancaster M, Hyatt A, van Driel R, Wong F, Warner S: Purification of a herpes-like virus from abalone (Haliotis spp.) with ganglioneuritis and detection by transmission electron microscopy.

    Journal of Virological Methods 2008, 149:338-341. PubMed Abstract | Publisher Full Text OpenURL

  4. Chang PH, Kuo ST, Lai SH, Yang HS, Ting YY, Hsu CL, Chen HC: Herpes-like virus infection causing mortality of cultured abalone Haliotis diversicolor supertexta in Taiwan.

    Dis Aquat Organ 2005, 65:23-27. PubMed Abstract | Publisher Full Text OpenURL

  5. Corbeil S, Colling A, Williams LM, Wong FYK, Savin K, Warner S, Murdoch B, Cogan NOI, Sawbridge TI, Fegan M, et al.: Development and validation of a TaqMan PCR assay for the Australian abalone herpes-like virus.

    Dis Aquat Organ 2010, 91:1-10. PubMed Abstract | Publisher Full Text OpenURL

  6. Davison AJ, Eberle R, Ehlers B, Hayward GS, McGeoch DJ, Minson AC, Pellett PE, Roizman B, Studdert MJ, Thiry E: The order Herpesvirales.

    Arch Virol 2009, 154:171-177. PubMed Abstract | Publisher Full Text OpenURL

  7. McGeoch DJ, Rixon FJ, Davison AJ: Topics in herpesvirus genomics and evolution.

    Virus Research 2006, 117:90-104. PubMed Abstract | Publisher Full Text OpenURL

  8. Waltzek TB, Kelley GO, Alfaro ME, Kurobe T, Davison AJ, Hedrick RP: Phylogenetic relationships in the family Alloherpesviridae.

    Dis Aquat Organ 2009, 84:179-194. PubMed Abstract | Publisher Full Text OpenURL

  9. Le Deuff RM, Renault T: Purification and partial genome characterization of a herpes-like virus infecting the Japanese oyster, Crassostrea gigas.

    J Gen Virol 1999, 80:1317-1322. PubMed Abstract | Publisher Full Text OpenURL

  10. Silander K, Saarela J: Whole genome amplification with Phi29 DNA polymerase to enable genetic or genomic analysis of samples of low DNA yield.

    Methods Mol Biol 2008, 439:1-18. PubMed Abstract | Publisher Full Text OpenURL

  11. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation.

    Bioinformatics 2000, 16:944-945. PubMed Abstract | Publisher Full Text OpenURL

  12. Farley CA, Banfield WG, Kasnic G Jr, Foster WS: Oyster herpes-type virus.

    Science 1972, 178:759-760. PubMed Abstract | Publisher Full Text OpenURL

  13. Davison AJ, Trus BL, Cheng N, Steven AC, Watson MS, Cunningham C, Le Deuff RM, Renault T: A novel class of herpesvirus with bivalve hosts.

    J Gen Virol 2005, 86:41-53. PubMed Abstract | Publisher Full Text OpenURL

  14. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

    Nucleic Acids Res 1997, 25:3389-3402. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  15. Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, Kawashima T, Robinson-Rechavi M, Shoguchi E, Terry A, Yu JK, et al.: The amphioxus genome and the evolution of the chordate karyotype.

    Nature 2008, 453:1064-1071. PubMed Abstract | Publisher Full Text OpenURL

  16. Davison AJ: Channel catfish virus: a new type of herpesvirus.

    Virology 1992, 186:9-14. PubMed Abstract | Publisher Full Text OpenURL

  17. Yang K, Homa F, Baines JD: Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus.

    J Virol 2007, 81:6419-6433. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  18. Zdobnov E, Apweiler R: InterProScan - an integration platform for the signature-recognition methods in InterPro.

    Bioinformatics 2001, 17:847-848. PubMed Abstract | Publisher Full Text OpenURL

  19. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.

    Nucleic Acids Res 1994, 22:4673-4680. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  20. Lartillot N, Philippe H: Improvement of molecular phylogenetic inference and the phylogeny of Bilateria.

    Philos Trans R Soc Lond B Biol Sci 2008, 363:1463-1472. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  21. Nielsen C: Six major steps in animal evolution: are we derived sponge larvae?

    Evol Dev 2008, 10:241-257. PubMed Abstract | Publisher Full Text OpenURL

  22. Arzul I, Renault T, Lipart C, Davison AJ: Evidence for interspecies transmission of oyster herpesvirus in marine bivalves.

    J Gen Virol 2001, 82:865-870. PubMed Abstract | Publisher Full Text OpenURL

  23. Arbuckle JH, Medveczky MM, Luka J, Hadley SH, Luegmayr A, Ablashi D, Lund TC, Tolar J, De Meirleir K, Montoya JG, et al.: The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

    Proc Natl Acad Sci USA 2010, 107:5563-5568. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  24. 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. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  25. Suttle CA: Viruses in the sea.

    Nature 2005, 437:356-361. PubMed Abstract | Publisher Full Text OpenURL

  26. Ehlers B, Dural G, Yasmum N, Lembo T, de Thoisy B, Ryser-Degiorgis MP, Ulrich RG, McGeoch DJ: Novel mammalian herpesviruses and lineages within the Gammaherpesvirinae: cospeciation and interspecies transfer.

    J Virol 2008, 82:3509-3516. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  27. Enquist LW, Husak PJ, Banfield BW, Smith GA: Infection and spread of alphaherpesviruses in the nervous system.

    Adv Virus Res 1998, 51:237-347. PubMed Abstract | Publisher Full Text OpenURL

  28. Terry LA, Stewart JP, Nash AA, Fazakerley JK: Murine gammaherpesvirus-68 infection of and persistence in the central nervous system.

    J Gen Virol 2000, 81:2635-2643. PubMed Abstract | Publisher Full Text OpenURL

  29. Sakarya O, Armstrong KA, Adamska M, Adamski M, Wang IF, Tidor B, Degnan BM, Oakley TH, Kosik KS: A post-synaptic scaffold at the origin of the animal kingdom.

    PLoS One 2007, 2:e506. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  30. Richards GS, Simionato E, Perron M, Adamska M, Vervoort M, Degnan BM: Sponge genes provide new insight into the evolutionary origin of the neurogenic circuit.

    Curr Biol 2008, 18:1156-1161. PubMed Abstract | Publisher Full Text OpenURL

  31. Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, Salamov A, Terry A, Shapiro H, Lindquist E, Kapitonov VV, et al.: Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization.

    Science 2007, 317:86-94. PubMed Abstract | Publisher Full Text OpenURL

  32. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.

    Mol Biol Evol 2007, 24:1596-1599. PubMed Abstract | Publisher Full Text OpenURL