A novel class of herpesvirus with bivalve hosts

Andrew J. Davison1, Benes L. Trus2,3, Naiqian Cheng3, Alasdair C. Steven3, Moira S. Watson1, Charles Cunningham1, Rose-Marie Le Deuff4,{dagger} and Tristan Renault4

1 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
2 Imaging Sciences Laboratory, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA
3 Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA
4 Laboratoire de Génétique et Pathologie, IFREMER, 17390 La Tremblade, France

Correspondence
Andrew J. Davison
a.davison{at}vir.gla.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Ostreid herpesvirus 1 (OsHV-1) is the only member of the Herpesviridae that has an invertebrate host and is associated with sporadic mortality in the Pacific oyster (Crassostrea gigas) and other bivalve species. Cryo-electron microscopy of purified capsids revealed the distinctive T=16 icosahedral structure characteristic of herpesviruses, although the preparations examined lacked pentons. The gross genome organization of OsHV-1 was similar to that of certain mammalian herpesviruses (including herpes simplex virus and human cytomegalovirus), consisting of two invertible unique regions (UL, 167·8 kbp; US, 3·4 kbp) each flanked by inverted repeats (TRL/IRL, 7·6 kbp; TRS/IRS, 9·8 kbp), with an additional unique sequence (X, 1·5 kbp) between IRL and IRS. Of the 124 unique genes predicted from the 207 439 bp genome sequence, 38 were members of 12 families of related genes and encoded products related to helicases, inhibitors of apoptosis, deoxyuridine triphosphatase and RING-finger proteins, in addition to membrane-associated proteins. Eight genes in three of the families appeared to be fragmented. Other genes that did not belong to the families were predicted to encode DNA polymerase, the two subunits of ribonucleotide reductase, a helicase, a primase, the ATPase subunit of terminase, a RecB-like protein, additional RING-like proteins, an ion channel and several other membrane-associated proteins. Sequence comparisons showed that OsHV-1 is at best tenuously related to the two classes of vertebrate herpesviruses (those associated with mammals, birds and reptiles, and those associated with bony fish and amphibians). OsHV-1 thus represents a third major class of the herpesviruses.

The GenBank/EMBL/DDBJ accession number of the OsHV-1 DNA sequence reported in this paper is AY509253.

SDS-PAGE analysis of the OsHV-1 capsid proteins is available as supplementary material in JGV Online.

{dagger}Present address: 9 rue Traversière, 17200 St Sulpice de Royan, France.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Viruses are assigned to the family Herpesviridae on the basis of morphological criteria and have been identified in a wide range of vertebrates and one invertebrate, the Pacific oyster, Crassostrea gigas (Minson et al., 2000). The vertebrate herpesviruses fall into two major phylogenetic groups. Those in the first group have mammalian or avian hosts and are classified into three subfamilies (Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae) that share extensive genetic relationships (McGeoch et al., 2000). Reptilian herpesviruses probably also belong among the Alphaherpesvirinae (Nigro et al., 2004; Quackenbush et al., 1998; Une et al., 2000; Yu et al., 2001). Viruses in the second group infect amphibians or bony fish and again are interrelated (Bernard & Mercier, 1993; Davison, 1998; Davison et al., 1999). Genetic evidence for a common evolutionary origin for the two groups is tenuous, however, since not a single herpesvirus-specific gene is detectably conserved in both. The only completely sequenced lower vertebrate herpesvirus, channel catfish virus (CCV), does share a few genes with the higher vertebrate group, but these have counterparts in other organisms (Davison, 1992). The conserved gene that comes closest to being herpesvirus specific encodes the putative ATPase subunit of the terminase, an enzyme complex involved in packaging viral DNA into preformed capsids. However, the presence of a distantly related gene in bacteriophage T4 leaves open the possibility of convergent evolution, with the two herpesvirus lineages having acquired this function independently. Nonetheless, similarities between the two vertebrate herpesvirus groups in capsid structure (Booy et al., 1996) and mechanisms of capsid maturation (Davison & Davison, 1995) tip the balance of evidence in favour of a common origin.

Particles morphologically similar to herpesviruses were first reported in an invertebrate (the Eastern oyster, Crassostrea virginica) by Farley et al. (1972) from the USA. Infections were detected contemporaneously in the UK in another oyster species, Ostrea edulis (D. J. Alderman, personal communication). Herpes-like viruses have since been identified in various marine bivalve species throughout the world, including the Pacific oyster, C. gigas (Hine et al., 1992; Nicolas et al., 1992; Renault et al., 1994b), European flat oyster, O. edulis (Comps & Cochennec, 1993; Renault et al., 2000a), Antipodean flat oyster, Ostrea angasi (Hine & Thorne, 1997), Chilean oyster, Tiostrea chilensis (Hine, 1997; Hine et al., 1998), carpet shell clam, Ruditapes decussatus (Renault & Arzul, 2001), Manila clam, Ruditapes philippinarum (Renault et al., 2001) and great scallop, Pecten maximus (Arzul et al., 2001a). Infections are often associated with sporadic episodes of high mortality among larvae and juveniles (Renault, 1998; Renault et al., 1994a, 1995). PCR-based diagnostic methods (Arzul et al., 2002; Barbosa-Solomieu et al., 2004; Lipart & Renault, 2002; Renault & Arzul, 2001; Renault et al., 2000a, b) have facilitated epidemiological investigations, for example showing that healthy adult animals can harbour the viral genome. Transmission experiments have demonstrated the virulence of the virus (Le Deuff et al., 1994) and indicated that a single species is probably responsible for all the infections observed (Arzul et al., 2001b, c). The ability of the virus to cause disease in a wide range of bivalve species may indicate that it has emerged as a consequence of selection under intensive farming conditions (Arzul et al., 2001c). The earliest genetic evidence for its presence in bivalves was obtained by in situ hybridization analysis of samples of O. edulis collected in 1976 in the UK (R.-M. Le Deuff, unpublished data).

The virus isolated from infected C. gigas larvae has been classified as a member of the Herpesviridae under the name ostreid herpesvirus 1 (OsHV-1) (Minson et al., 2000). Initial characterization yielded an estimated genome size of 180 kbp, and a small (266 bp) fragment demonstrated no sequence similarity to other herpesviruses (Le Deuff & Renault, 1999). The purpose of the present work was to analyse the capsid morphology and genome sequence of OsHV-1 in order to assess its relationship to vertebrate herpesviruses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Purification of OsHV-1 capsids.
Frozen infected C. gigas larvae (0·5–1·0 ml packed volume) that were PCR-positive for OsHV-1 were ground with an equal volume of sand in 9 ml NTE (0·5 M NaCl, 20 mM Tris/HCl, pH 7·5, 2 mM EDTA) by using a pestle and mortar maintained on ice. Triton X-100 [10 % (v/v) in NTE] was added to a final concentration of 1 % (v/v) and the mixture was probe sonicated on ice for 1 min. Capsids were prepared essentially as described by Davison & Davison (1995), with all centrifugation steps carried out in a Sorvall TST-41 rotor at 4 °C. The sonicated material was centrifuged for 10 min at 10 000 r.p.m. (maximum 17 000 g). The supernatant was placed in a fresh tube and underlayed with 1 ml 40 % (w/v) sucrose in NTE and centrifuged for 1 h at 20 000 r.p.m. (maximum 68 000 g). The pellet was resuspended in 0·5 ml NTE and centrifuged on a linear 10–40 % (w/v) sucrose gradient in NTE for 1 h at 20 000 r.p.m. The capsid band was visualized using illumination from a light pipe and isolated by side puncture of the tube, diluted in 10 ml PBS and pelleted for 1 h at 20 000 r.p.m. The capsids were resuspended in approximately 10 µl PBS and frozen and transported on dry ice. A second batch was prepared similarly, except that the concentration of NaCl in NTE was reduced to 0·05 M in order to reduce ionic disruption and the capsids were transported on wet ice.

Cryo-electron microscopy of OsHV-1 capsids.
Cryo-electron microscopy was performed as described by Newcomb et al. (2000) using an FEI CM-200 FEG electron microscope operating at 120 kV and low-electron-dose methods. Seven micrographs, recorded at a magnification of x38 000 and digitized on a Perkin-Elmer 1010MG microdensitometer at 25 µm per pixel, yielded a total of 248 particle images at 6·6 Å (0·66 nm) per pixel. These data were analysed and a reconstruction calculated using the polar Fourier transform (PFT) model-based procedure to determine orientations and origins (Baker & Cheng, 1996). A previously calculated map of herpes simplex virus type 1 (HSV-1) A-capsids (Cheng et al., 2002) was used as a starting model. After four cycles of PFT refinement, a stable three-dimensional reconstruction was calculated from 161 images to a resolution of 25 Å (0·25 nm).

Isolation of OsHV-1 virion DNA.
Cell-culture methods for growth of OsHV-1 do not exist. Consequently, OsHV-1 DNA was extracted from virions purified from infected C. gigas larvae obtained from a commercial hatchery (Le Deuff & Renault, 1999).

Cloning of OsHV-1 DNA fragments.
Cosmid libraries were prepared by inserting OsHV-1 DNA partially digested with BamHI or Sau3AI into the BamHI site of a derivative of Supercos 1 (Stratagene) (Cunningham & Davison, 1993) and packaging the products into bacteriophage {lambda} particles using a Stratagene III XL kit. A total of 320 cosmids was analysed by BamHI digestion.

A primary bacteriophage {lambda} library of OsHV-1 BamHI fragments (5x104 recombinants) was produced using the Lambda ZAP II vector (Stratagene) and a Stratagene III Gold packaging kit. The amplified library was subjected to the manufacturer's mass excision protocol to derive a plasmid library. A total of 84 plasmids was assessed by BamHI digestion.

A plasmid library of OsHV-1 BamHI fragments inserted directly into the BamHI site of the pUC18 vector was generated. A total of 288 plasmids was characterized by BamHI digestion. Attempts were made to clone several fragments absent from this and the excised {lambda} libraries by purifying OsHV-1 BamHI fragments from an agarose gel and inserting them into pUC18. The resulting clones were analysed by digestion with appropriate restriction endonucleases, using genome sequence data to predict cleavage patterns. The identities of inserts in the final plasmid library were confirmed by DNA sequencing.

Analysis of OsHV-1 genome structure.
Restriction endonuclease and Southern blot hybridization analyses (the latter employing 32P-labelled DNA probes) were carried out using standard procedures.

Sequencing of the OsHV-1 genome.
An M13mp19 library of random OsHV-1 DNA fragments was sequenced using an ABI PRISM 377 instrument. The sequence database was compiled from electropherograms using Pregap4 and Gap4 (Staden et al., 2000) and Phred (Ewing & Green, 1998; Ewing et al., 1998). Regions of ambiguity were resolved by sequencing PCR products directly or as plasmid clones. The sequence was determined to a mean redundancy of 10·8, and 96·1 % was obtained on both strands. The purity of the OsHV-1 DNA preparation, as judged from the number of M13 clones whose sequence did not match the database, was 99 %.

The genome sequence was reconstructed from the database after locating the genome termini, which were predicted approximately from restriction endonuclease profiles and mapped experimentally as described by Davison et al. (2003). This involved ligating a partially double-stranded adaptor to flush-ended OsHV-1 DNA and carrying out PCR using an adaptor-specific primer plus a primer specific for the left or right terminal region of the genome (5'-GAGTGCCCAGGTGCATTATG-3' or 5'-CACGGCCGTTGTATAGGAGG-3', respectively). The junction between forms of the terminal sequences present internally in the genome was amplified using the two OsHV-1-specific primers. Products were cloned as plasmids and sequenced.

The genome sequence was analysed using locally implemented forms of the GCG (Accelrys), CLUSTAL W (Thompson et al., 1994) and Ptrans programs (Taylor, 1986) and online versions of the BLAST (Altschul et al., 1997), FastA (Pearson & Lipman, 1988), SignalP v2.0 (Nielsen et al., 1997) and PRED-TMR2 programs (Pasquier & Hamodrakas, 1999).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Capsid structure of OsHV-1
Despite husbanding OsHV-1 capsids in small volumes, the low yields rendered sparse fields of particles when prepared for cryo-electron microscopic observation over holey carbon films. Nevertheless, by searching for relatively well-populated areas and combining data from multiple micrographs, sufficient images were accumulated to yield a reconstruction at a resolution of approximately 25 Å (0·25 nm).

OsHV-1 capsids were similar in overall appearance to those of other herpesviruses that have been studied to date (Booy et al., 1996; Cheng et al., 2002; Trus et al., 1999, 2001). The diameter estimated from cryo-electron microscopic images (approx. 116 nm) was slightly less than that of HSV-1 capsids (125 nm). This is somewhat surprising in view of the larger genome size (207 kbp compared with 152 kbp), but the DNA could be accommodated if packaged to the same density as in human cytomegalovirus (HCMV) (Bhella et al., 2000). Most particles were roughly hexagonal in outline, corresponding to views close to three- or twofold axes of symmetry (Fig. 1). The striking feature was the double layer of density around the peripheries. In this respect, they differed from other herpesvirus capsids, which tend to have serrated peripheries generated by external protrusions seen in side-view. The reconstruction shown in Fig. 2(a–c) provides an explanation for this difference, which arose because the OsHV-1 triplexes extended radially almost as far outwards as the hexon protrusions. The double layer of peripheral density was occasionally interrupted by low-density patches (Fig. 1).



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Fig. 1. Cryo-electron micrographs of individual OsHV-1 capsids. The arrowheads denote patches of low peripheral density due to the absence of pentons. Bar, 50 nm.

 


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Fig. 2. Cryo-electron microscopic reconstruction of the OsHV-1 capsid viewed along a twofold axis of symmetry: (a) outside surface, (b) inside surface and (c) central section. The arrows indicate density corresponding to the triplexes. For comparison, HSV-1 capsids are shown: (d) lacking pentons owing to treatment with guanidine hydrochloride (G-capsid; reproduced from Newcomb et al., 1993, by permission of Elsevier) and (e) an untreated reference (A-capsid). For further comparison, (f) shows the capsid of CCV (reproduced from Booy et al., 1996, by permission of Elsevier). The OsHV-1 capsid is slightly smaller (by approx. 7 %) than that of HSV-1. Bar, 50 nm.

 
Reconstruction of the OsHV-1 capsid (Fig. 2a–c) revealed an icosahedral structure with a triangulation number of T=16. This confirmed that OsHV-1 was a herpesvirus, since this surface lattice geometry has been observed only with members of this family. A most striking feature was the large holes at the vertices, marking the absence of pentons from these sites. These holes explained the low-density patches seen on the original images (Fig. 1). Observations of HSV-1 have shown that pentons are more susceptible to proteolytic degradation than hexons (Trus et al., 1996) and pentons may be extracted selectively by treatment with denaturants at concentrations that leave the rest of the surface lattice intact (Fig. 2d and e) (Newcomb et al., 1993). There is other evidence that HSV-1 pentons are conformationally different from hexons (Cheng et al., 2002). Unlike hexons, they do not bind the small capsid protein VP26 (Booy et al., 1994), and certain monoclonal antibodies differentiate between the penton and hexon conformations of the 150 kDa major capsid protein VP5 (Trus et al., 1992). Another example of pentons representing the weakest points in an icosahedral shell is provided by their heat-induced loss from mature bacteriophage P22 capsids (Teschke et al., 2003). The OsHV-1 capsids were not exposed to denaturants or heat, and the holes were observed in both batches studied. By analogy with known properties of other herpesviruses, we supposed that 11 of the OsHV-1 vertices were originally occupied by pentons of the major capsid protein and the twelfth by a portal protein complex, but that OsHV-1 vertex proteins are more selectively labile than those of HSV-1 and were degraded or dislodged at some point during the isolation procedure. The reconstructed OsHV-1 capsid was similar to that of HSV-1 with its pentons extracted (Fig. 2d) and, taking into account the absence of pentons, to the intact HSV-1 and CCV capsids (Fig. 2e and f).

Other features of the OsHV-1 capsid were also distinctively herpesvirus-like. These included prominent external protrusions at the hexon sites, rising above the continuous floor region, and the relatively flat and featureless appearance of the inner surface. Because the facets were rather flat, not round, it was likely that the particles analysed were derived from mature capsids rather than precursor procapsids. Masses corresponding to triplexes were present at all sites of local threefold symmetry (best seen at a glancing angle and in cross-section; upper arrows in Fig. 2b and c). In other previously characterized herpesvirus capsids, triplexes are heterotrimers consisting of two copies of a protein of about 35 kDa and one copy of another protein of approximately 50 or 35 kDa, depending on the virus (Trus et al., 2001). The triplexes initially play a morphogenic role in the assembly of the procapsid and then, in the mature capsid, switch to a structural role as clamps that stabilize the joints between groups of three neighbouring capsomeres (Steven & Spear, 1997).

Many higher vertebrate herpesviruses have a small capsid protein (10–15 kDa; VP26 in HSV-1) that caps the hexons but not the pentons and whose presence may be inferred from comparing hexon and penton structures (Wingfield et al., 1997). Such a comparison was not possible with the preparations of OsHV-1 capsids and thus the question of whether OsHV-1 has a corresponding protein remains unanswered. However, we noted the presence of a protein of appropriate size among those generated by SDS-PAGE of OsHV-1 capsids (see supplementary Fig. 1, available at JGV Online). These proteins included species of 150 kDa (presumably the major capsid protein) and 14 kDa, plus less-abundant species of 40, 32 and 27 kDa.

Genome structure of OsHV-1
The BamHI profile of OsHV-1 DNA is shown in Fig. 3, alongside a list indicating the order of fragments along the genome and the identities of fragments that were cloned as cosmids or plasmids. Two of the five fragments that were not cloned as plasmids were terminal (A, L) and would not be expected to be represented. The locations of the other three uncloned fragments (R, I and E), plus the minimum size requirement for a cosmid insert (35 kbp), had the effect of excluding from the cosmid library five intervening fragments (U, H, V, S and P) that were obtained as plasmids. Two regions of the genome, in fragments R and E, were also not represented in the M13 library, necessitating direct sequencing of PCR products. The sequence features presumably responsible for failure to clone certain regions of the genome were not readily apparent.



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Fig. 3. Sizes of OsHV-1 BamHI fragments and bacterial clones obtained. The BamHI profile of OsHV-1 DNA is shown to the left, with each fragment indicated by a letter and size (bp) derived from the genome sequence. The upper and lower portions were obtained from 0·6 and 1·5 % (w/v) agarose gels, respectively, stained with ethidium bromide. Fragments containing the left (L) and right (S) genome termini are indicated. Four small fragments (Z, a, b and c) detected in other experiments are not visible in these gels. To the right, the BamHI fragments are listed as arranged along the genome from left to right, with estimated sizes (derived by agarose gel electrophoresis) and actual sizes (calculated from the genome sequence). Dark-grey shading shows fragments cloned successfully into cosmids (BamHI and Sau3AI libraries with inserts of 35–45 kbp containing several contiguous BamHI fragments) or plasmids (containing individual BamHI fragments). Light-grey shading shows fragments from which portions were present in cosmids (at the ends of inserts in the Sau3AI library).

 
The structure of the sequence database implied that the genome contained two unique regions (UL and US; 167 843 and 3370 bp, respectively), each flanked by an inverted repeat (TRL/IRL and TRS/IRS; 7584 and 9774 bp, respectively), with the internal copies of the repeats separated by a third unique region (X; 1510 bp). Thus, the genome may be represented as TRL–UL–IRL–X–IRS–US–TRS. The total genome size was 207 439 bp, close to the sum of the estimated sizes of BamHI fragments (Fig. 3) and larger than the value published initially (180 kbp; Le Deuff & Renault, 1999). The nucleotide composition was 38·7 mol% G+C.

The 5' ends of the genome were not unique, but a predominant form was apparent for each (Fig. 4). PCR of the region between IRL and IRS yielded two products (data not shown), the larger containing X, as expected, and the smaller lacking it. The latter would correspond to a ligated form of the termini if the 3' end of each terminus possessed unpaired nucleotides complementary to those at the other (Fig. 4), as is the case for vertebrate herpesviruses (Mocarski & Roizman, 1982). In experiments in which probes from X and flanking regions of IRL and IRS were hybridized to HindIII fragments of OsHV-1 DNA (Fig. 5a), each probe detected the 7645 bp fragment containing X and adjacent portions of IRL and IRS. The IRL probe also detected the 1245 bp terminal fragment from TRL (electrophoresed from the gel in Fig. 5a, but confirmed in other experiments) and the IRS probe detected the 4890 bp terminal fragment from TRS. In addition, the X and IRL probes hybridized to a 2755 bp fragment of low abundance, which represented the terminal fragment from TRL joined to a copy of X. However, a fragment corresponding to the fused form of the genome termini with X lacking (6135 bp) was not detected. These results indicated that the major genome form is as described above, that a small proportion of molecules contains a copy of X at the left genome end and that an even smaller proportion may lack the internal copy of X.



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Fig. 4. Sequences of the OsHV-1 genome termini in comparison with the junction in a minority of molecules that corresponds to a fusion of the termini (i.e. the major junction lacking X). The sequences of the termini have been inverted and complemented so that they align with the junction. All sequences are shown as a single 5'->3' strand. A tilde (~) indicates the location of contiguous sequence, which is not shown, and the terminal nucleotides are underlined. The number of clones obtained for each sequence is shown to the left. The possibility that the junction was an artefact generated by priming at one terminus by the other, utilizing unpaired nucleotides, rather than having originated from genomes lacking X internally, is rendered unlikely by the small number of unpaired nucleotides potentially present (0–9 at each terminus, with a mode of 2).

 


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Fig. 5. Data confirming the OsHV-1 genome structure. Standard size markers were included in all gels, but are not shown. All fragment sizes (bp) were derived from the genome sequence. (a) Autoradiograph showing the results of hybridizing radiolabelled probes to a HindIII digest of OsHV-1 DNA transferred from a 0·6 % (w/v) agarose gel. The probes were OsHV-1 DNA and regions from X (nt 184202–184416, as a PCR product excised from a plasmid) and IRL and IRS immediately adjacent to X (nt 182634–183011 and 184522–184878, respectively, as PCR products). A longer exposure of the lower half of the first lane has been superimposed and a longer exposure of the result obtained using the X probe (third lane) is provided in the second lane. (b) Ethidium bromide-stained 0·6 % (w/v) agarose gel showing larger ClaI fragments of OsHV-1 DNA, with sizes of submolar bands from the genome termini and the IRL–IRS junction indicated. (c) Autoradiograph showing the results of hybridizing radiolabelled probes to a ClaI digest of OsHV-1 DNA transferred from a 0·6 % (w/v) agarose gel. The probes were OsHV-1 DNA and cloned PCR products from the left and right ends of UL (nt 7586–7785 and 175227–175426, respectively) and the left and right ends of US (nt 194324–194539 and 197461–197662, respectively). The correspondence with fragments shown in (b) is indicated. (d) Ethidium bromide-stained 0·7 % (w/v) agarose gel showing fragments in the KpnI profile of OsHV-1 DNA. Major and minor fragments diagnostic of inversion of a 4·8 kbp region in UL are indicated.

 
Vertebrate herpesvirus genomes containing two unique regions flanked by large inverted repeats, such as those of HSV-1 and HCMV, exist in virions as four isomers differing in the relative orientations of the unique regions (Hayward et al., 1975; Weststrate et al., 1980). As a consequence, certain restriction endonucleases produce ‘half-molar’ or ‘quarter-molar’ fragments from the IRL–IRS junction and half-molar fragments from one or both genome termini, depending on whether they cleave the inverted repeats. The sizes of fragments produced by digestion of OsHV-1 DNA with a variety of restriction endonucleases showed that genome isomerization was a property of OsHV-1, and their abundance indicated that virion DNA contained approximately equimolar amounts of the four isomers. For example, ClaI, which cleaves UL and US but neither repeat, produced four half-molar fragments of 10 256–12 421 bp and four quarter-molar fragments of 22 262–25 319 bp (Fig. 5b). Hybridization experiments using probes from the ends of UL and US confirmed the identities of these fragments, each probe detecting terminal and junction fragments of appropriate sizes (Fig. 5c). Thus, probes from the left and right ends of UL and the left and right ends of US hybridized to terminal fragments of 11 388, 10 256, 10 496 and 12 421 bp, respectively, and also to pairs of junction fragments (the components largely indistinguishable from each other in Fig. 5c) of 25 319/23 394, 24 187/22 262, 23 394/22 262 and 25 319/24 187 bp, respectively.

A further complexity in genome structure was revealed by several of the M13 clones used to construct the sequence database. These implied that a minor population of genomes existed with an inversion of a 4·8 kbp region in the centre of UL (green bar in Fig. 6). This was supported by the detection of minor restriction endonuclease fragments whose sizes were consistent with them containing the two junctions between the inverting region and flanking sequences in the minor population. For example, both minor (8514 and 15 003 bp) and major fragments (10 494 and 13 023 bp) were detected in the KpnI profile (Fig. 5d). The proportions indicated that approximately 25 % of the DNA population contained the 4·8 kbp region in inverse orientation.



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Fig. 6. Layout of genes in the OsHV-1 genome. The scale is in kbp and the ‘ORF’ prefix is omitted throughout. The inverted repeats TRL/IRL (ORF1–ORF6) and TRS/IRS (ORF116–ORF122) are shown in a thicker format than the unique regions UL (ORF6–ORF114), US (ORF123–ORF124) and X (ORF115). By reference to the key, coloured bars above the genome denote features in the DNA sequence described in the text and coloured arrows in the genome indicate protein-coding regions grouped according to the key. Square brackets apply to genes whose fragmented protein-coding regions are depicted as intact.

 
The preparations of OsHV-1 DNA examined therefore contained a mixture of genome forms. Some were an inherent property of the genome structure and others probably reflect the fact that the DNA originated from a virus that had not been clonally purified. The major structure, TRL–UL–IRL–X–IRS–US–TRS, was present by virtue of inversion of UL and US as four isomers in approximately equimolar amounts. Some molecules contained an additional copy of X at the left terminus and some might have lacked X internally. This could represent X–TRL–UL–IRL–X–IRS–US–TRS, perhaps with X–TRL–UL–IRL–IRS–US–TRS and TRL–UL–IRL–IRS–US–TRS, each again as four isomers. Since herpesvirus genomes are considered to be packaged into capsids from head-to-tail concatemers, some of these minor forms might result from rare cleavage of concatemers at the X–TRS, rather than IRL–IRS, junction. Some genomes also contained a 4·8 kbp region within UL that was inverted. Presumably, this population also consisted of the various permutations described above.

Genetic content of OsHV-1
In order to identify potential protein-coding regions in the genome (Fig. 6), an initial set of open reading frames (ORFs) was defined, in which each ORF consisted of at least 100 codons and was flanked by termination codons. Smaller ORFs overlapping larger ones by more than 60 % of their length were then discounted, except for ORF17 and ORF18, which overlapped slightly larger ORFs on the opposing strand. These were retained because each encoded a putative membrane protein and ORF17 was followed by a potential polyadenylation signal. One ORF of 117 codons was discounted because it spanned the X–RS junction, had an unusually high G+C content and lacked a potential polyadenylation signal. Three additional ORFs (ORF16, ORF36 and ORF37) smaller than 100 codons were then added to the set. ORF16 and ORF36 did not overlap other ORFs and encoded putative membrane proteins, and ORF37 overlapped ORF38 to a small extent. In order to represent protein-coding regions, the 5' end of each ORF was trimmed to the first ATG codon, except for ORF120, where use of the second ATG gave rise to a strong prediction for a signal sequence at the N terminus of the encoded protein. The analysis indicated the presence of 124 unique protein-coding genes (Fig. 6). Owing to the presence of the inverted repeats, 12 ORFs were duplicated, resulting in a total of 136 genes in the genome. These numbers included several fragmented genes (see below), each of which was counted as a single ORF. All ORFs were followed by a potential polyadenylation signal or were a member of a potential 3'-coterminal set. No indications of splicing emerged from sequence analysis.

One striking property of the gene complement was the presence of 38 genes in 12 families of related genes (Fig. 6). These included two families whose products were predicted to be secreted (ORF13 with two members and ORF50 with four), three families predicted to encode membrane-associated proteins (ORF41 with three members, ORF54 with two and ORF88 with four), one family whose products contained motifs V and VI of SF2 helicases (ORF7 with three members; motifs defined by Gorbalenya & Koonin, 1993), one family whose products were related to inhibitors of apoptosis (BIR with four members), one family derived from a deoxyuridine triphosphatase gene [(dut) with three members, of which probably only ORF75 encoded an active enzyme; active site motifs defined by McGeoch, 1990], two families of RING-finger genes (RING-1 with four members and RING-2 with two) and two other families (ORF4 with five members and ORF35 with two). The ancient nature of the duplication events that have resulted in these families was illustrated by the fact that family members were distantly related and generally widely distributed in the genome. Gene families are also prevalent in vertebrate herpesviruses and numerous in the larger genomes (Chee et al., 1990).

The BIR family had four members (ORF42, ORF87, ORF99 and ORF106) that encoded products belonging to a family of viral and cellular proteins known as ‘baculovirus inhibitor of apoptosis repeat proteins' (BIRPs), several of which have been shown to have anti-apoptotic activity (reviewed by Miller, 1999). The ORF42 and ORF106 proteins also contained a RING finger. BIRP-regulated pathways are evidently important mediators of defence against viral infection in invertebrates, since BIR genes are found not only in OsHV-1 but also in other large DNA viruses with invertebrate (insect) hosts, including members of the Baculoviridae (Crook et al., 1993), Ascoviridae (Stasiak et al., 2000), Poxviridae (Afonso et al., 1999), Iridoviridae (Jakob et al., 2001) and Asfarviridae (Yáñez et al., 1995). In contrast, large DNA viruses of vertebrates do not encode BIRPs and subvert host apoptotic defences by other means.

Another characteristic of the OsHV-1 genome was the presence of disrupted genes in certain of the gene families (ORF5 in the ORF41 family; ORF32, ORF63 and ORF65 in the ORF32 family; and ORF50, ORF62, ORF73 and ORF105 in the ORF50 family). Although the disrupted genes contained more than one ORF, they were counted as single entities in calculating the OsHV-1 gene complement and Fig. 6 shows the locations of the intact coding regions that presumably existed before fragmentation occurred. The genes involved were detected from amino acid sequence alignments as between two and five tandem ORFs arranged in different reading frames or separated by termination codons, but similar to successive regions in relatives that were fragmented differently or not at all. Most or all are presumably non-functional and it was notable that no intact member of the ORF50 family and only one intact member of the ORF32 family remained. The degree of divergence between sequences generally precluded identifying precisely the locations and, in some cases, the number of fragments. As an example, Fig. 7 shows details of the proposed disintegration of ORF5 in relation to its intact relatives. There are four fragments, separated by two frameshifts and one termination codon. It was notable that the ORF5 protein also lacked a C-terminal region that was present in the related proteins and contained a putative transmembrane domain. This raised the possibility that ORF5 has been affected by deletion of this region. Moreover, ORF5 was more extensively deleted in an OsHV-1 variant (orange bar in Fig. 6; Arzul et al., 2001c; the end nearer UL was not mapped precisely). The ability to align sequences within gene families was essential for detecting disrupted genes and it cannot be ruled out that genes not belonging to families may also be disrupted. Gene fragmentation has not been observed in vertebrate herpesviruses, but is a characteristic of many members of the Poxviridae (reviewed by Gubser et al., 2004). It is not possible to discern at present whether apparent loss of gene function in OsHV-1 reflects the selection of a pathogenic variant from a genetically intact ancestor, which may have been more species restricted and benign, as a result of modern large-scale shellfish farming activities.



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Fig. 7. Amino acid sequence alignment of ORF41 and ORF59 with sequences that make up ORF5, an apparently disrupted member of the gene family. The four separate coding regions that comprise ORF5 are shown in different colours. The first three are in different reading frames and the third and fourth are in the same reading frame but separated by a termination codon (asterisk). The ends of these regions are identified with a degree of uncertainty, but are contiguous with respect to the DNA sequence (i.e. there are no gaps). Predicted signal and transmembrane sequences are underlined near the N and C termini of the proteins, respectively. The consensus (con) is shown in upper case for residues that are identical in all three proteins and in lower case for residues that are identical in two.

 
The gene layout in the 4·8 kbp region within UL that is inverted in some genomes (green bar in Fig. 6) was difficult to assess, since it contained all or part of three disrupted genes. The interpretation supported from alignments was that the minor form (utilized in Fig. 6) was the parent of the major form. In the latter, ORF62 and ORF65, both of which were disrupted, are present as fragmented genes representing hybrids between the ORF50 and ORF88 families. This complex rearrangement was presumably arrived at by a series of recombination and mutation events whose order and effect are obscure.

Database searches provided functional information on 25 OsHV-1 genes that were not members of families. These included genes encoding a tentative primase (ORF24; based on a short motif defined by Klinedinst & Challberg, 1994), two subunits of ribonucleotide reductase (ORF20 and ORF51), a helicase (ORF67), the catalytic subunit of a DNA polymerase {delta} (ORF100), the ATPase subunit of terminase (ORF109; Davison, 2002) and two RING-like proteins (ORF53 and ORF124). The ORF95 protein was related to a family of conserved eukaryotic proteins (e.g. the FLJ21144protein in humans), which are also more distantly related to the nuclease domain in bacterial RecB proteins (Aravind et al., 1999; Chang & Julin, 2001). The ORF30 protein was related in an N-terminal cysteine-rich domain to a protein of unknown function in two subfamilies (Beta- and Gammaherpesvirinae) of the higher vertebrate herpesviruses (e.g. UL92 in HCMV and ORF31 in human herpesvirus 8). The consensus of this domain is CX20–21CX2CX3HXCX5CX6–10CX3G. A total of 15 genes encoded proteins that had predicted signal or transmembrane sequences and therefore may be associated with membranes, one (ORF57) specifying a protein that was related to a cellular chloride ion channel.

The OsHV-1 sequence contained several families of imperfect repeats, ranging in size from 3 to 146 bp (red bars in Fig. 6). Many were present as a single copy at each locus, and all but one were intergenic. Also, a large palindrome (Fig. 8) was located between ORF49 and ORF50 (blue bar in Fig. 6), most of which was deleted upon cloning into plasmids. By analogy with higher vertebrate herpesviruses in the subfamily Alphaherpesvirinae (Stow & McMonagle, 1983; Weller et al., 1985) and the Roseolovirus genus of the subfamily Betaherpesvirinae (Inoue et al., 1994), the palindrome is a candidate for an origin of DNA replication.



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Fig. 8. Structure of the intact OsHV-1 palindrome in viral DNA and its deleted form in plasmids. Base pairing within one of the two DNA strands is shown. Non-Watson–Crick base paired nucleotides within the paired regions are shown in bold. The regions between which the deletion occurred in plasmids are indicated by horizontal or vertical lines on the viral sequence.

 
Evolution and taxonomy of OsHV-1
Despite the similarities in capsid structure between OsHV-1 and the two vertebrate herpesvirus groups, sequence comparisons did not reveal any OsHV-1 proteins with convincing similarity to known capsid proteins of either group. Moreover, global comparisons failed to identify OsHV-1 genes that had homologues only in other herpesviruses, with the exception of ORF30, which had a domain found to date elsewhere only in a subset of mammalian herpesviruses but not in CCV. ORF109, which encoded the putative ATPase subunit of the OsHV-1 terminase, had relatives in all vertebrate herpesviruses and in T4-related bacteriophages. The OsHV-1 and T4 genes are not spliced, whereas those in higher and lower vertebrate herpesviruses contain one and two introns, respectively. In principle, phylogenetic analysis of ORF109 and its counterparts should reveal the branching order, but it was not possible to draw a conclusion beyond that of the three groups being highly diverged. Thus, although a scheme may be proposed in which OsHV-1 and the two vertebrate herpesvirus groups descended from a common ancestor with divergence so extensive that sequence similarity is not detectable, the evidence must be regarded as scant. Nonetheless, it is clear that herpesviruses fall into three major classes, one associated with mammals, birds and reptiles, one with amphibians and bony fish, and one with bivalves. The logical means of establishing this in formal taxonomy would be to establish three virus families incorporating the three groups as listed above, under the umbrella of an order (Herpesvirales).


   ACKNOWLEDGEMENTS
 
This work was supported by EC Contract FAIR-CT98-4334 (VINO). We are grateful to Kathleen Wright and Aidan Dolan for technical assistance and to Duncan McGeoch for comments on the manuscript.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 19 June 2004; accepted 30 September 2004.