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1 Department of Veterinary Pathology, University of Edinburgh, Edinburgh, UK
2 Institute of Virology, University of Zurich, Zurich, Switzerland
3 Division of Medical Microbiology, School of Infection and Host Defence, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, UK
4 Division of Virology, Moredun Research Institute, Edinburgh, UK
Correspondence
James P. Stewart
j.p.stewart{at}liv.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY839756.
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), where losses can be substantial. The disease is mainly caused by either of two closely related bovid gammaherpesviruses (
HVs), Alcelaphine herpesvirus 1 (AlHV-1) and Ovine herpesvirus 2 (OvHV-2), that persist subclinically in their natural host. These viruses are related closely in biological properties and sequence to each other and to a recently identified virus, porcine lymphotropic herpesvirus 1 (PLHV-1), that causes post-transplant lymphoproliferative disease in pigs (Goltz et al., 2002). OvHV-2 and AlHV-1 are related more distantly to other HVs, such as herpesvirus saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein–Barr virus (EBV) and Murid herpesvirus 4 (MuHV-4). AlHV-1 naturally infects wildebeest (Connochaetes spp.) and is the source of wildebeest-associated MCF in Africa (Plowright et al., 1960). OvHV-2 is endemic in domestic sheep (Ovis aries), which act as a reservoir of infection for the other main form of the disease, sheep-associated MCF (SA-MCF) (Baxter et al., 1993; Li et al., 1998; Müller-Doblies et al., 1998; Wiyono et al., 1994). Aside from sporadic outbreaks in domestic cattle, SA-MCF is the most important virus disease of farmed deer and has recently been reported in pigs (Albini et al., 2003; Loken et al., 1998). SA-MCF is also currently a disease of great concern in Indonesia, affecting Bali cattle, and in the USA, where bison are particularly susceptible (Li et al., 2006; O'Toole et al., 2002; Schultheiss et al., 2000).
In the reservoir species, sheep, OvHV-2 DNA has been found by PCR in B cells in the bloodstream, lymph nodes and the respiratory, alimentary and urogenital tracts (Baxter et al., 1997; Hüssy et al., 2002). OvHV-2 DNA has also been detected in nasal and ejaculatory secretions, suggesting possible respiratory- and sexual-transmission mechanisms (Hüssy et al., 2002; Li et al., 2004). Moreover, cattle have been infected experimentally with nasal secretions from infected sheep, showing that respiratory transmission is likely (Taus et al., 2006). In contrast, in SA-MCF-affected ruminants, virus DNA is usually detected by PCR in lymph nodes and spleens (Müller-Doblies et al., 2001) and has been observed by in situ hybridization in hyperplastic T cells in brain lesions (Simon et al., 2003). Thus, to enable the study of the interaction of OvHV-2 with host cells, T-lymphoblastoid cell lines with the morphology of large granular lymphocytes (LGLs) have been established in culture from the tissues of MCF-affected animals (Reid et al., 1983, 1989; Schock & Reid, 1996; Swa et al., 2001). These T-cell lines contain OvHV-2 DNA and antigen (Baxter et al., 1993; Bridgen & Reid, 1991; Swa et al., 2001) and can transmit MCF experimentally to rabbits and hamsters (Buxton et al., 1984, 1988), which are used as animal models. OvHV-2-positive LGLs generally have a T-cell phenotype, are constitutively and indiscriminately (non-major histocompatibility complex-restricted) cytotoxic and produce a range of cytokines, but not interleukin-2 (IL-2) (Schock & Reid, 1996; Schock et al., 1998; Swa et al., 2001). Our current hypothesis is that MCF is due to indiscriminate tissue damage caused by dysregulated cytotoxic T cells generated as a consequence of infection. The LGL T cells in culture represent the virus-infected cells in vivo and are invaluable for virus–cell interaction studies in MCF.
AlHV-1 has been isolated, will infect epithelial cell lines productively in culture and has been sequenced completely (Ensser et al., 1997; Plowright et al., 1960). In contrast, research on OvHV-2 has lagged behind, due to the lack of a productive tissue-culture system and reagents. This work describes the complete sequence of the OvHV-2 genome as a first step in the detailed molecular analysis of SA-MCF. An accompanying manuscript by Taus et al. (2007) describes a comparison of our sequence with that of OvHV-2 derived from the nasal secretions of sheep.
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METHODS |
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). This line was maintained in Iscove's modified Dulbecco's medium supplemented with 2 mM glutamine (Invitrogen), 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 10 % heat-inactivated fetal bovine serum and 350 IU IL-2 ml–1 (Proleukin; Chiron Therapeutics).
Construction of a cosmid library.
A cosmid library containing OvHV-2 DNA was generated from BJ1035 cells by using Supercos1 (Stratagene), modified as described by Cunningham & Davison (1993) (a kind gift of Dr A. Davison, MRC Virology Unit, University of Glasgow, Glasgow, UK). High-molecular-mass DNA extracted from BJ1035 cells was digested partially with MboI such that the products were on average 40 kb in size. A cosmid library was then generated in Supercos1 from the MboI-digested BJ1035 DNA as described in the Supercos1 manual (Stratagene). Briefly, cosmid arms generated from the vector by restriction-enzyme digestion and dephosphorylation were ligated to MboI-cut BJ1035 DNA. Ligated DNA was packaged into recombinant 
phage by using a Gigapack III XL packaging kit (Stratagene) according to the manufacturer's instructions. The library was then amplified once using Escherichia coli XL-1 Blue MR and stored at –80 °C.
Screening cosmid library.
Bacterial colonies from the cosmid library were screened by colony hybridization at high stringency (Sambrook et al., 1989) using probes of known OvHV-2 sequence. DNA probes of 300–500 bp in length were generated by PCR amplification using either DNA extracted from OvHV-2-infected cell lines or cosmid DNA as a template. DNA probes were labelled with [
-32P]dCTP by using a random-primed DNA labelling kit (Roche). Positive colonies underwent a second round of screening. Cosmid DNA was prepared from colonies positive on the second screen by using QIAspin mini prep kits (Qiagen). The ends of the inserted DNA were then sequenced by using the vector-specific primers 5'-AAGGAAACGACAGGTGCTG and 5'-CGAAAATGTCCACCTGACGTC, which lie either side of the insert sites in the modified Supercos1.
DNA sequencing.
DNA sequencing was performed by using the dideoxy chain-termination sequencing method. Sequencing of cosmid ends, splinkerette products and plasmids containing the terminal repeat elements was performed by using either the in-house sequencing service at the Department of Veterinary Pathology, University of Edinburgh, UK, or via Lark Technologies.
Sequencing of the four overlapping cosmid clones was performed by a shotgun library approach using pCR4bluntTopo (Invitrogen). Plasmid subclones were cycle-sequenced with BigDye Terminator version 1.0 reagents (Applied Biosystems) and analysed on a MegaBACE 1000 sequencer (Amersham Biosciences) or an ABI 377 sequencer (Applied Biosystems). Computer-assisted assembly was done with Lasergene SeqMan (DNASTAR Inc.) with five- to sevenfold redundancy.
PCR amplification.
PCRs of 50 µl total volume contained 1x PCR buffer [20 mM Tris (pH 8.4), 50 mM KCl; Invitrogen], 1.5 mM MgCl2, 250 µM each of dATP, dGTP, dCTP and dTTP (Ultrapure dNTP set; Amersham Biosciences), 200 pmol each primer, 100–500 ng DNA template and 1 U Taq DNA polymerase (Invitrogen). PCR primers were obtained from MWG-Biotech. PCR programs generally consisted of 30–40 cycles of 30 s denaturing at 94 °C, 1 min annealing at 55–60 °C and extension at 72 °C for 1 min (kb product)–1. To generate PCR products across the genome termini, a GC-rich PCR kit (Roche Diagnostics) was used in combination with primers homologous to the ends of the known sequence. The genome co-ordinates of the primers were as follows: sense, 128577–128598; antisense, 467–487.
Analysis of OvHV-2 gene splicing by RT-PCR.
Total RNA was isolated from BJ0135 cells by extraction using RNeasy kits (Qiagen) according to the manufacturer's guidelines, digested with RQ1 DNase (Promega) (0.1 U µl–1) for 30 min at 37 °C, extracted sequentially in phenol/chloroform and chloroform and then precipitated in ethanol. cDNA was synthesized from 2 µg total RNA with SuperScript II reverse transcriptase (Invitrogen) primed with an oligo(dT) adapter primer (Gibco-BRL) in a 20 µl reaction according to the manufacturer's recommendations. Aliquots (1 µl) of cDNA were then amplified by PCR using primers specific for OvHV-2 ORFs. Amplified cDNAs were then analysed by gel electrophoresis, inserted into the cloning vector pCR2.1-TOPO and multiple clones for each cDNA were sequenced. The genome co-ordinates for primers used for RT-PCR are as follows: Ov2, sense 2813–2792, antisense 2162–2183; Ov2.5, sense 3576–3597, antisense 4455–4434; Ov6, sense 79355–79375, antisense 80327–80307; ORF57, sense 89060–89072, antisense 90482–90460; Ov8, sense 81538–81560, antisense 83906–83886; Ov8.5, sense 117777–117796, antisense 118950–118930.
Genome walking by splinkerette PCR.
Splinkerette PCR is a method of extending from known to unknown sequence by amplification of DNA sequences that lie between a single known primer and a nearby restriction site (Devon et al., 1995). Splinkerette PCR was performed on BJ1035 DNA exactly as described by Devon et al. (1995). Oligonucleotide adapters specific for the enzymes BamHI, HindIII, SalI and EagI were utilized. PCR products generated were cloned by using a pCR2.1-TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions. Multiple clones (five to seven) from each walk were sequenced.
Nucleotide and protein sequence analysis.
The EMBOSS (Rice et al., 2000) package of programs was used for final assembly and analysis of sequences, including gene finding. The definition of open reading frames (ORFs) was performed by using the GeneMarkS program with the eukaryotic virus option (Besemer et al., 2001), as well as by comparison with other herpesviruses and mammalian genes using the NCBI BLAST programs (Altschul et al., 1990; Gish & States, 1993). Annotation was performed by using the Artemis program (Rutherford et al., 2000).
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RESULTS AND DISCUSSION |
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The cloning and sequencing strategy is shown in Fig. 1 and the co-ordinates of relevant clones are detailed in Table 1. A cosmid library was made by using total high-molecular-mass DNA from BJ1035 cells. An initial OvHV-2 cosmid (c75) was isolated by using a probe consisting of part of ORF75 (Bridgen & Reid, 1991). Cosmid clones corresponding to a large part of the unique portion of the genome were then isolated successively by using probes derived from the ends of the cosmid inserts. The DNA sequence of these cosmids was determined after shotgun cloning into pCR4bluntTopo and sequencing. Despite repeated attempts, we were unable to isolate further cosmid clones that spanned or contained genome termini. To complete the genome sequence, successive splinkerette PCRs were performed, walking away from the known sequence. However, upon reaching high G+C content and repetitive sequence, corresponding to terminal repeat elements (TRs), we were unable to proceed any further with this technique.
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; Thonur et al., 2006), PCR amplification using primers homologous to the ends of the known sequence and a GC-rich PCR kit (Roche Diagnostics) was used to generate independent plasmid clones that completed a circular sequence. Analysis of several independent clones (represented by p3.5) revealed that they were identical in sequence and that there was a GC-rich region of approximately 600 bp within each clone. As the TRs of HVs are GC-rich, we surmised that this could correspond to one copy or part of the TR element. However, it was impossible to determine the boundaries of the unique region and TR by using this clone. Analysis revealed a unique HindIII site in the centre of the 600 bp GC-rich region in p3.5. To determine the exact nature of the TR unit, BJ1035 DNA was therefore cut with HindIII and analysed by Southern blotting using p3.5 as a probe (not shown). This revealed a hypermolar fragment of approximately 4.2 kbp in length, corresponding to one TR unit. The 4.2 kbp HindIII fragment was subsequently cloned into the vector pBluescript KS+ (Stratagene). A number of clones with the same sequence, containing one unit of the TR (pH4.2), were isolated. Comparison of the sequences of p3.5 and pH4.2 allowed the definition of the boundaries of the unique and TR DNA and additionally showed that the PCR fragment in p3.5 was generated from a circular, defective OvHV-2 genome containing only a single, deleted TR unit. The boundaries of the TR and unique DNA were further confirmed by PCR analysis and sequencing.
In line with the convention for other HVs, the OvHV-2 sequence was assembled in the same orientation as that of HVS (Albrecht et al., 1992), with the sequence of one copy of the TR placed after the end of the unique sequence. The sequence of the unique region was 130 930 bp in length, with a mean G+C content of 52 mol%, and the sequence of one TR unit was 4205 bp in length, with a mean G+C content of 72 mol%.
Repeat regions in the unique portion of the OvHV-2 genome
In addition to the TRs, analysis of the unique portion of the genome using the EMBOSS programs EQUICKTANDEM and EINVERTED revealed six tandem- and two inverted-repeat structures. These are shown in Fig. 2 and detailed in the GenBank entry (accession no. AY839756
[GenBank]
). Five of the tandem-repeat elements are contained within coding regions, two within the unique ORF Ov8.5 and three within ORF73. Repeats within ORF73 homologues are also seen in related viruses, such as AlHV-1 and KSHV. Repeats at the same position as those within Ov8.5 are seen in a number of HVs [e.g. Bovine herpesvirus 4 (BoHV-4), MuHV-4 and KSHV] and are known to form part of the lytic origins of replication in these viruses (AuCoin et al., 2002; Deng et al., 2004; Lin et al., 2003; Zimmermann et al., 2001). Thus, this region of the genome may also act as a lytic origin in OvHV-2. The two inverted repeats are located in a long region of apparently non-coding DNA between ORFs 11 and 17.5. Although not conserved in sequence, these repeats are positionally analogous to two inverted repeats in the AlHV-1 genome (Ensser et al., 1997). They may therefore perform a conserved function, such as origin of DNA replication.
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; Table 2). Potential protein-encoding ORFs were identified by the following criteria: ORF size larger than 60 aa, presence of potential transcriptional start and stop sites, a high GeneMark score and homology to other known herpesvirus or cellular ORFs. In line with the nomenclature of other HVs, where applicable, ORFs were assigned the number of the homologue in HVS. When possible, ORFs with homologues shared only with AlHV-1 were assigned the same number as in AlHV-1, but with the Ov prefix for ovine. ORFs with no homologues in HVS or AlHV-1 were assigned an Ov prefix with numbers between the adjacent Ov ORFs.
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HVs. The genome structure, ORF content and similarity of amino acid sequence were closest to those of AlHV-1, with many AlHV-1 ‘unique’ ORFs also being found in OvHV-2. A comparison of the AlHV-1 and OvHV-2 genomes is shown in Fig. 3. Conserved OvHV-2 ORFs were arranged in four blocks collinear with other HVs, as indicated in Table 2 and Fig. 3.
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) and PLHV-1 (Goltz et al., 2002). The validity of all candidate OvHV-2-unique ORFs (those with an Ov prefix) has been confirmed, as mRNAs corresponding to all of these ORFs were detected in OvHV-2-infected cells (Thonur et al., 2006).
The left end of the unique region contains four ORFs. ORFs Ov2 and Ov3 are homologous to AlHV-1 A2 and A3, respectively. Ov2 contains two exons, as confirmed by RT-PCR and sequencing. It encodes a protein containing a basic leucine-zipper (bZIP) motif, is homologous to ATF-like, CREB and Jun dimerization proteins and is therefore likely to be a transcription factor. The product of Ov3 contains a putative signal peptide to residue 22 and, like AlHV-1 A3, is homologous to proteins of the semaphorin family. However, Ov3 is shorter than A3 by 200 aa at the C-terminus, the truncation occurring just after the consensus Sema domain (InterPro IPR001627). There is a short stretch of collinear homology between A3 and the region downstream of the stop codon for the Ov3 ORF. In addition, the Ov3 ORF has a long predicted 3' untranslated region, making the gene a similar length [predicted TATA to poly(A) signal] to the A3 gene. It seems likely, therefore, that the Ov3 ORF is the product of truncation of a longer ORF. The Ov3 protein shows the greatest similarity in amino acid sequence to semaphorin 7A (CDw108) and poxvirus semaphorins, e.g. vaccinia virus A39R. Semaphorin 7A is expressed by lymphoid and myeloid cells and, like A39R, is a potent monocyte activator (Comeau et al., 1998; Holmes et al., 2002). Thus, Ov3 may be involved in the modulation of the host response to OvHV-2.
There are no homologues of the AlHV-1 A1 and A4 ORFs in OvHV-2. Instead, there are two unique ORFs, Ov2.5 and Ov3.5. Ov2.5 contains five exons (as confirmed by RT-PCR and sequencing) and encodes a homologue of cellular IL-10. This ORF is interesting, in that it retains precisely the exon structure of the cellular gene, unlike other herpesvirus IL-10 homologues. Ov2.5 has also been shown to encode a functional IL-10 molecule that can block cytokine secretion by macrophages and stimulate proliferation of mast cells (J. P. Stewart & D. M. Haig, unpublished observations) and may thus be involved in the modulation of the host response to OvHV-2. In contrast, Ov3.5 has no significant homology to any known protein, encodes a peptide of 163 aa and contains a putative signal peptide. It is therefore likely to be secreted from the infected cell.
Between conserved ORFs 03 and 06 lies the Ov4.5 ORF. This encodes a protein with homology to the EBV BALF1, equine herpesvirus 2 E6 and cellular Bcl-2 proteins. Comparative sequence analysis of the AlHV-1 genome showed that a homologous ORF, now termed A4.5, was not reported in the published description of the sequence (Ensser et al., 1997). A second OvHV-2 ORF, Ov9, also encodes a Bcl-2 homologue. This ORF is situated at the right-hand end of the unique region and is homologous to AlHV-1 A9. Thus, like EBV, it appears that OvHV-2 (and AlHV-1) encodes two Bcl-2 family homologues. In EBV, BHRF1 is anti-apoptotic, whereas the role of BALF1 is controversial, being reported as both pro- and anti-apoptotic in transfected cell lines (Bellows et al., 2002; Marshall et al., 1999). Recent deletion analysis has, however, assigned an essential role for both proteins in EBV transformation of B cells by protecting newly infected cells from apoptosis prior to the establishment of latency. Thus, it is possible that, in OvHV-2-infected cells, the Ov4.5 and Ov9 products promote survival of infected lymphocytes and the establishment of latency.
The Ov5 ORF is located downstream of ORF9/DNA polymerase and overlaps with ORF10. This ORF is predicted to encode a G protein-coupled receptor (GPCR) that is homologous to the AlHV-1 A5, except that it is predicted to have a longer C-terminal (intracellular) tail than A5. Iteration of the PSI-BLAST program revealed that Ov5 is also related to EBV BILF1 and more weakly to cellular IL-8 receptors. BILF1 functions as a constitutively signalling (ligand-independent) GPCR that alters intracellular signalling (Beisser et al., 2005; Paulsen et al., 2005); hence, the Ov5 protein may fulfil a similar role in OvHV-2-infected cells.
OvHV-2, like AlHV-1 and PLHV-1, is unusual amongst HVs in that it has no ORF28 homologue. ORF28 encodes a non-essential virion glycoprotein in other HVs (Bortz et al., 2003; May et al., 2005). Thus, the function of ORF28 is either redundant or performed by a separate glycoprotein in this subgroup of HVs. However, unlike AlHV-1, OvHV-2 does encode an ORF49 homologue. The ORF49 homologue of EBV (BRRF1) has been shown to act as a transcriptional transactivator that co-operates with the viral BRLF1 transactivator (ORF50) to induce lytic replication (Hong et al., 2004). PLHV-1 also contains an ORF49 homologue, so AlHV-1 is highly unusual in not encoding a homologous ORF and its absence is not a consistent feature of this group of HVs.
In between ORF50 and ORF52 lie three ORFs, Ov6, Ov7 and Ov8. These all have homologues in AlHV-1 (A6, A7 and A8) and in PLHV-1. Like the PLHV-1 homologue, Ov6 was shown to consist of three exons, as determined by RT-PCR analysis and sequencing. The product of Ov6 contains a leucine-zipper motif in its C-terminal region and there are consensus DNA-binding motifs towards the N terminus. It also has significant sequence similarity to the CCAAT/enhancer-binding protein family, consistent with it having a putative role in transcriptional transactivation. In addition, although there is no direct sequence relationship, Ov6 is positionally analogous to EBV BZLF1 and KSHV K8, which are both transactivators of the viral lytic cycle. It seems likely, therefore, that Ov6 may fulfil a similar function during OvHV-2 infection. The product of Ov7 contains a predicted signal peptide and N-linked glycosylation motifs and is thus likely to be a viral glycoprotein. Although there is no sequence similarity, Ov7 is positionally analogous to EBV BZLF2, whose product is involved in entry of EBV into B cells via binding to HLA-DR (Spriggs et al., 1996). The Ov7 protein may also therefore be involved in receptor binding. Ov8 was shown to consist of two exons by RT-PCR and sequencing. These splice sites are conserved in the homologous ORFs in AlHV-1 and PLHV-1 and correspond to the regions of homology between these proteins. Thus, the AlHV-1 A8 and PLHV-1 A8 may be spliced in a similar fashion. The product of Ov8 was predicted to contain a transmembrane anchor near the C terminus and N-linked glycosylation sites and so, like Ov7, is likely to be a virus glycoprotein. Also, like Ov7, although there is no sequence similarity, Ov8 is positionally analogous to EBV BLLF1 (gp350/220), KSHV K8.1 and MuHV-4 ORF51, all of which encode glycoproteins involved in binding to cellular receptors (Birkmann et al., 2001; Stewart et al., 2004; Tanner et al., 1987). Thus, the Ov8 protein may also be involved in binding to cellular receptors.
Downstream of ORF73 lies Ov8.5, which is unique to OvHV-2 and shows no obvious similarity to any viral or cellular gene. Ov8.5 is predicted to encode a proline-rich (24 %) peptide of molecular mass 42 kDa that contains no consensus motifs, as defined by the PROSITE database. However, RNA from Ov8.5 is found in OvHV-2-infected cells (Thonur et al., 2006) and, so, this is a bone fide ORF. The N-terminal region of the Ov8.5 protein is encoded by two DNA direct-repeat elements. Direct-repeat elements that form part of the origins of virus DNA replication are present in the analogous genomic location in other HVs, e.g. BoHV-4, KSHV and MuHV-4 (AuCoin et al., 2002; Deng et al., 2004; Lin et al., 2003; Zimmermann et al., 2001). In BoHV-4 and KSHV, unique ORFs (Bo11, Bo12 and K12) are also found surrounding the repeat. Thus, the presence of a direct repeat and unique ORF at a genomic location analogous to that of Ov8.5 appears to be a common feature of HVs. Further studies are required to show whether this region acts as an origin of DNA replication and to assign a function to Ov8.5.
Directly upstream of Ov8.5 is ORF73. This shows significant similarity to ORF73 of other HVs, including KSHV. However, the similarity is largely restricted to the C-terminal region. Like other homologues, OvHV-2 ORF73 incorporates a large acidic-repeat domain. Variability in the length of the acidic-repeat domain is seen between isolates of KSHV and HVS ORF73 proteins (Ensser et al., 2003; Gao et al., 1999; Zhang et al., 2000). In an accompanying study, Taus et al. (2007) show that ORF73 derived from the nasal secretions of sheep varied from the sequence reported here in the length of the acidic domain. It is not clear what functional difference this variability makes. However, differences in ORF73 sequence may be useful for epidemiological studies. The KSHV ORF73 was described as a latency-associated nuclear protein (LANA) that functions to transactivate the viral latent origin of replication (Hu et al., 2002). It seems likely, therefore, that the OvHV-2 homologue will have a similar function.
The final unique ORF found was Ov10. This lies at the right-hand end of the unique region between Ov9 and the terminal repeats. This ORF shows limited similarity to AlHV-1 A10. The predicted Ov10 protein has a potential transmembrane anchor at the C terminus and four consensus nuclear-localization signals. Thus, the Ov10 protein may localize to the nucleus of infected cells.
Conserved spliced ORFs
The region encoding ORF40/41 in OvHV-2 consisted of one continuous ORF with regions that were homologous to ORFs 40 and 41 of other herpesviruses. These ORFs are conserved amongst all herpesviruses and encode a protein that is complexed with helicase and primase. In many HVs, such as EBV (Fixman et al., 1995) and KSHV (AuCoin & Pari, 2002; Wu et al., 2001), the coding sequence for this protein is formed by splicing of two separate ORFs, 40 and 41. In other HVs, such as MuHV-4, there is a continuous ORF, but this is still spliced at conserved splice sites (J. P. Stewart, unpublished observations). The sequence of ORF40/41 of OvHV-2 contained conserved splice sites that corresponded to regions of homology to other HV ORFs 40 and 41. It seems likely, therefore, that OvHV-2 ORF40/41 is spliced in a fashion similar to that of MuHV-4. Interestingly, the OvHV-2 sequence derived from the nasal secretions of sheep, described by Taus et al. (2007) in a complementary study, contains an additional 2 nt in the predicted intron in ORF40/41, resulting in two separate ORFs. To confirm the sequence of the BJ1035 virus, PCR products across the region were generated from BJ1035 cellular DNA, sequenced and found to be identical to the original sequence. Thus, within the OvHV-2 species, there are variants with either a single or separate ORFs 40/41. However, this difference is likely to be silent, as the predicted final transcripts are identical.
The transcripts for viral terminase (ORF29), transcriptional transactivator (Rta/ORF50) and ORF57 are known to be formed from the splicing of two exons in other HVs. Consensus potential splice-donor and -acceptor sites for these OvHV-2 homologues (Table 2) were present and determined by comparative sequence analysis.
Similarity to other MCF-associated viruses
It has been proposed that the HVs associated with MCF be placed in their own genus, Macavirus (McGeoch et al., 2006), based on evolutionary relatedness of conserved ORF sequences. The sequence presented here confirms this new grouping, showing that OvHV-2 is highly similar to AlHV-1 and PLHV-1, not only in the nucleotide similarity of conserved ORFs, but also in terms of ORFs that are present only in this group of viruses. These macavirus-specific ORFs are likely to be involved in host-specific pathogenesis and the development of MCF. Thus, comparative genetic analysis of OvHV-2 and related viruses enabled by the completion of this sequence will be core to the understanding of the mechanisms underlying MCF.
The sequence of OvHV-2 derived from the nasal secretions of sheep, published in a complementary study by Taus et al. (2007), shows that, whilst the two genomes are extremely similar, there are differences. As the outbreaks of MCF in European cattle are sporadic in nature, it has been hypothesized that they could be due to the generation of more pathogenic OvHV-2 variants. Analysis of the sequence differences between the nasal-secretion virus (derived from the reservoir species) and the sequence derived from a clinically affected cow (BJ1035) will be important to determine whether these are relevant to pathogenicity in cattle.
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ACKNOWLEDGEMENTS |
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Received 9 June 2006;
accepted 19 September 2006.
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). Subsequent cosmids were isolated by using probes derived from the cosmid ends. Sequences to the end of the unique portion of the genome and the terminal repeats were derived by a series of splinkerette walks and, finally, cloning of the terminal repeats into a plasmid as described in the text.
) DNA strands. Major repetitive elements are shown as shaded rectangles, direct repeats as hashed blocks and inverted repeats as solid bars.