Novel herpesviruses of Suidae: indicators for a second genogroup of artiodactyl gammaherpesviruses

Bernhard Ehlers1 and Stewart Lowden2

1 P11/Neuartige Viren, Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany
2 Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK

Correspondence
Bernhard Ehlers
ehlersb{at}rki.de


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Five novel herpesviruses were identified in suid species from Africa (common warthog, Phacochoerus africanus) and South-East Asia (bearded pig, Sus barbatus; babirusa, Babyrousa babyrussa) by detection and analysis of their DNA polymerase genes. Three of the novel species, P. africanus cytomegalovirus 1, P. africanus lymphotropic herpesvirus 1 (PafrLHV-1) and S. barbatus lymphotropic herpesvirus 1 (SbarLHV-1), were closely related to known beta- (porcine cytomegalovirus) and gammaherpesviruses [porcine lymphotropic herpesvirus (PLHV) 1 and 3] of domestic pigs. In contrast, two novel species, S. barbatus rhadinovirus 1 (SbarRHV-1) and Babyrousa babyrussa rhadinovirus 1 (BbabRHV-1), were more closely related to a ruminant gammaherpesvirus, bovine herpesvirus 4 (BoHV-4), than to the porcine gammaherpesviruses PLHV-1, -2, -3, PafrLHV-1 and SbarLHV-1. SbarRHV-1, BbabRHV-1 and BoHV-4 were therefore tentatively assigned to a novel genogroup of artiodactyl gammaherpesviruses. This latter genogroup may also contain an as yet undiscovered gammaherpesvirus of domestic pigs, thereby adding a concern to their use in xenotransplantation.


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Domestic pigs (Sus scrofa) have been proposed as organ donors for xenotransplantation. A significant potential hazard and safety issue in xenotransplant patients is the transmission of porcine viruses (Auchincloss & Sachs, 1998; O'Connell et al., 2000; Onions et al., 2000). Porcine herpesviruses are of particular concern since human herpesviruses, predominantly cytomegalovirus and Epstein–Barr virus (EBV), are associated with severe disease in allotransplantation (Ferry & Harris, 1994; van Zanten et al., 1998). Furthermore, herpesviruses are often inapparent in their natural hosts, but result in severe disease after infection of foreign hosts. To illustrate, the B herpesvirus causes only a benign infection in rhesus monkeys but induces fatal encephalitis in humans (Eberle & Hilliard, 1995). Another example is ovine herpesvirus 2 (OvHV-2). This virus causes only a benign infection in sheep, yet results in a fatal malignant catarrhal fever in cattle and pigs (Albini et al., 2003). Consequently, considerable efforts are under way to produce experimental donor pigs free of a number of known swine microorganisms (Fishman, 1994; Tucker et al., 2002).

We recently reported the discovery of three porcine gammaherpesviruses in S. scrofa. Due to their lymphotropism, they were named porcine lymphotropic herpesviruses 1, 2 and 3 (PLHV-1, -2, -3). PLHV-1 and -3 and PLHV-2 have been found to be highly prevalent in domestic and feral pig populations, respectively (Ehlers et al., 1999b; Ulrich et al., 1999; Chmielewicz et al., 2003a). Evidence for the pathogenic potential of PLHV-1 was obtained using a porcine model of haematopoietic stem cell transplantation. This virus induces a fatal lymphoproliferative disorder resembling human post-transplant lymphoproliferative disease (PTLD), which occurs predominantly after liver and bone marrow allotransplantation (Huang et al., 2001). While human PTLD is frequently associated with EBV, porcine PTLD has been found to be associated with PLHV-1 (Goltz et al., 2002).

Genetic analyses provide additional evidence for potential pathogenicity within the PLHVs. Together with several ruminant gammaherpesviruses (of cattle, sheep, goats and deer), PLHVs form a distinct genogroup within the Gammaherpesvirinae (Chmielewicz et al., 2003a). Some of these ruminant viruses (alcelaphine herpesvirus 1 and OvHV-2) are associated with malignant catarrhal fever (MCF), a systemic, usually fatal, lymphoproliferative disorder of artiodactyla. MCF occurs after transmission to foreign hosts, while the natural hosts of these viruses are only latently infected (Heuschele, 1988; Reid & Buxton, 1989; Crawford et al., 1998; Albini et al., 2003).

Based on this evidence, gammaherpesviruses are of major concern when pigs are used as organ donors for xenotransplantation. Nucleic acid-based (Ehlers et al., 1999b; Chmielewicz et al., 2003a) and antibody-based (S. Brema, M. Goltz & B. Ehlers, unpublished) detection methods were therefore developed to allow monitoring of the PLHV status of donor pigs and xenotransplanted patients. Furthermore, domestic pigs were extensively analysed for the presence of additional, yet unknown, porcine herpesviruses, but such viruses were not found. From this work, we tentatively concluded that no further herpesviruses were present in the domestic pig population (Chmielewicz et al., 2003b).

Here, we have followed an indirect strategy to detect still undiscovered herpesviruses of domestic pigs. Other species within the family Suidae were analysed for the presence of herpesviruses. With this potential for detecting new herpesviruses, we also had the opportunity to analyse domestic pigs retrospectively for these viruses.

Blood and tissue samples of Suidae from Africa [common warthog (Phacochoerus africanus) n=31] and South-East Asia [babirusa (Babyrousa babyrussa), n=22; Western bearded pig (Sus barbatus oi), n=8; Bornean bearded pig (Sus barbatus barbatus), n=9; and Sulawesi warty pig (Sus celebensis), n=12] were analysed with a pan-herpes PCR assay for the herpesviral DNA polymerase (DPOL) gene. DPOL was targeted by nested PCR using degenerate primers DFA, ILK and KG1 in the first round and TGV and IYG in the second round of amplification (described by Ehlers et al., 1999a; modified by Chmielewicz et al., 2001). Suitable amplimers were purified and sequenced (see Goltz et al., 2002, for methods). Sequences of 166, 175 and 178 bp (excluding primer binding sites) were obtained, the length depending upon the herpesvirus species. When novel sequences were detected, specific primer pairs were designed and retested in the suid species of origin. In a parallel experiment, domestic pigs were also analysed for the presence of these sequences.

From the 82 samples tested, 54 were positive using the pan-herpes PCR. In addition to known porcine herpesviruses, sequence analysis also revealed the presence of novel beta- and gammaherpesviruses. The viruses are listed in Table 1 with their tentative names and GenBank accession numbers.


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Table 1. Novel suid beta- and gammaherpesviruses

 
PLHV-1 DPOL sequences (sharing 100 % nucleic acid identity) were found in 6/8 S. barbatus oi and 3/9 S. barbatus barbatus from a South-East Asian zoo, 3/12 wild-caught S. celebensis from Sulawesi and 9/31 P. africanus from Zimbabwe. PLHV-3 DPOL sequences (sharing 100 % nucleic acid identity) were found in 4/22 B. babyrussa from both European and South-East Asian zoos. A novel sequence with 98 % nucleic acid (100 % amino acid) identity to PLHV-3 DPOL was found in 4/8 S. barbatus oi. Notably, in 3/24 and 5/24 wild-caught P. africanus from Zimbabwe, novel sequences were found, which revealed 86 and 82 % amino acid identity to DPOL of PLHV-3 and porcine cytomegalovirus (PCMV; Goltz et al., 2000), respectively (Fig. 1). The viruses from which these three sequences originated were tentatively named SbarLHV-1, PafrLHV-1 and PafrCMV-1, respectively (Table 1). We interpreted these findings as showing that viruses identical, or similar, to PLHV-1, PLHV-3 and PCMV are commonly present in suid species from Africa and Asia.



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Fig. 1. Multiple sequence alignments of the novel suid beta- and gammaherpesvirus DNA polymerases. Nucleic acid sequences of beta- (b) and gammaherpesviruses (a) were translated and aligned with ClustalW (Lasergene, MegAlign module). Identical amino acids are represented by dots. Gaps are represented by dashes. Column 1 (AA) represents the length of the determined amino acid sequence. Columns 2–5 (at the far right) represent percentages of pairwise amino acid identity (%id). The names of the novel viruses are written in bold and marked with black arrowheads. Sequences of known herpesviruses were taken from GenBank. The accession numbers of the gammaherpesviruses are given in the legend to Fig. 2; those of the betaherpesviruses are: cytomegalovirus (CMV, X17403); human herpesvirus 6 (HHV-6, X83413); human herpesvirus 7 (HHV-7, U43400); PCMV (porcine cytomegalovirus, AF268042).

 
Surprisingly, in 2/8 S. barbatus oi and 12/22 B. babyrussa, two novel and distinct gammaherpesviruses were detected, which revealed, in pairwise amino acid comparisons, only 42–44 % identity to the PLHV species found in domestic pigs (PLHV-1, -2, -3) and the PLHV-like species found in S. barbatus oi and P. africanus (SbarLHV-1 and PafrLHV-1) (Fig. 1). The highest percentages of identity (70 and 75 %, respectively) were found with a ruminant gammaherpesvirus, the bovine herpesvirus 4 (BoHV-4), which is a member of the genus Rhadinovirus (Zimmermann et al., 2001). The viruses from which these two sequences originated were therefore tentatively named SbarRHV-1 and BbabRHV-1, respectively (Table 1). SbarRHV-1 and BbabRHV-1 still revealed a 10–12 % closer identity to the human herpesvirus 8 and the type species of the genus Rhadinovirus [herpesvirus saimiri (HVS), or saimirine herpesvirus 2] than to the PLHVs and the PLHV-like SbarLHV-1 and PafrLHV-1. Furthermore, the SbarRHV-1 and BbabRHV-1 sequences (55 aa) were identical in length to the corresponding DPOL sequences of BoHV-4, HHV-8 and HVS but not to those of PLHV-1, -2, -3, SbarLHV-1 and PafrLHV-1 (59 aa) (Fig. 1).

To confirm these data with analyses of extended sequences, we amplified and sequenced a 4 kb and 1·8 kb region of SbarLHV-1 using PLHV-3-specific primers. In PLHV-3, the 4 kb segment encompassed the 3'-end of the glycoprotein B (gB) gene (ORF8), the complete DNA polymerase gene (ORF9) and the 5'-part of a gene (A5/BILF1h) encoding a putative G-protein-coupled receptor (GCR). The 1·8 kb segment was non-coding and was located between ORFs 11 and 17 (for an ORF map, see Chmielewicz et al., 2003a). Between the gB, DPOL and GCR genes of SbarLHV-1 and PLHV-3, respectively, 97 (94), 98 (99) and 96 (94) % nucleic acid (amino acid) identities were found. The intergenic regions were 95 % identical. In contrast, the intraspecies nucleic acid identity of PLHV-3 was always more than 99·9 % in coding regions and more than 99·0 % in non-coding regions (Chmielewicz et al., 2003a). Furthermore, domestic pigs have only tested positive for PLHV-3 (Chmielewicz et al., 2003a). In this study, only SbarLHV-1 sequences, but not PLHV-3 sequences, were found in S. barbatus. Therefore, the SbarLHV-1 sequences appeared to originate from a distinct viral entity that exists only in S. barbatus and is not merely a strain of PLHV-3.

To extend the short DPOL sequences of SbarRHV-1, BbabRHV-1 and PafrLHV-1 in the upstream direction, hemi-nested PCR was performed. The degenerate sense primer DFA and two virus-specific antisense primers were used, as described previously (Ehlers et al., 2003). For SbarRHV-1 and BbabRHV-1, DPOL consensus sequences of 472 bp were generated. However, sequence extension was repeatedly unsuccessful for PafrLHV-1, using two different sets of antisense primers. This failure was probably caused by a low viral copy number and/or insufficient binding of the degenerate primer DFA.

Sequence comparisons were then repeated using the 157 aa DPOL sequences of SbarRHV-1, BbabRHV-1 and those of other gammaherpesviruses (156–161 aa). SbarRHV-1 revealed 74, 67 and 59 % identity to BoHV-4, HHV-8 and PLHV-1, respectively. BbabRHV-1 revealed 75, 66 and 59 % identity levels to the same viruses. These identity levels, although somewhat higher, confirmed the results of the 55 aa comparisons.

Phylogenetic analyses were performed with a multiple amino acid alignment using the maximum-likelihood method of the Tree-Puzzle program (Schmidt et al., 2000) or the neighbour-joining method of the PHYLIP program package (Felsenstein, 1996). In accordance with the results of pairwise amino acid comparisons, SbarRHV-1 and BbabRHV-1 formed a clade with BoHV-4. This was distinct from PafrLHV-1 and SbarLHV-1, which formed a clade with PLHV-1, -2 and -3 and several gammaherpesviruses of ruminants. This was supported by high probability values (Fig. 2).



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Fig. 2. Phylogenetic analysis of the novel suid gammaherpesviruses. A phylogenetic tree was constructed using the DPOL sequences of the novel gammaherpesviruses (Table 1) and the following additonal gammaherpesviruses, available in GenBank: AlHV1 (alcelaphine herpesvirus 1, AF005370); BLHV (bovine lymphotropic herpesvirus; AF327830); BoHV4 (bovine herpesvirus 4, AF318573); CalHV3 (callithrichine herpesvirus 3, AF319782); CprHV2 (caprine herpesvirus 2, AF327829); EBV (Epstein–Barr virus, X00784); HHV8 (human herpesvirus 8, U93872); HVA (herpesvirus ateles=ateline herpesvirus 3, AF083424); HVS (herpesvirus saimiri=saimirine herpesvirus 2, X64346); OvHV2 (ovine herpesvirus 2, AF327831); PLHV1 (porcine lymphotropic herpesvirus 1, AF478169); PLHV2 (porcine lymphotropic herpesvirus 2, AY170317); PLHV3 (porcine lymphotropic herpesvirus 3, AY170316); RRV (rhesus monkey rhadinovirus, AF083501). A multiple alignment of 153 aa was analysed with the maximum-likelihood method of the program Tree-Puzzle (Version 5.0). A rooted phylogram is shown, with EBV as the outgroup. The branch length is proportional to evolutionary distance (scale bar). Support values, estimated by the quartet puzzling (QP) tree search and expressing the QP reliability as a percentage, are indicated at the nodes of the tree to the left of the vertical divider. In addition, the alignment was analysed with the neighbour-joining method of the PHYLIP program package (version 3.6). The tree topology was the same (not shown) and the results of bootstrap analysis (100-fold) are presented to the right of the vertical divider. The novel viruses are highlighted in bold type. The branch of PafrLHV-1 was derived from analysis of 55 aa sequences (not shown) and is indicated by a dotted line. Genogroups I and II of artiodactyl gammaherpesviruses and suborders of artiodactyla are marked.

 
In conclusion, based on the results of nucleotide and amino acid sequence comparisons (Fig. 1) and phylogenetic analyses (Fig. 2), we have put forward the hypothesis that two distinct genogroups of artiodactyl gammaherpesviruses exist. Genogroup I comprises the porcine lymphotropic herpesviruses PLHV-1, -2 and -3, as well as PafrLHV-1, SbarLHV-1 and several gammaherpesviruses of ruminants (cattle, sheep, goats, deer), while genogroup II comprises SbarRHV-1, BbabRHV-1 and BoHV-4.

It was observed in this study that PLHV, which had been previously reported to infect domestic pigs, also infected B. babyrussa (PLHV-3), S. barbatus (PHLV-1) and P. africanus (PLHV-1). Therefore, we also tested domestic pigs (S. scrofa) for the presence of the novel suid viruses, particularly those of genogroup II (BbabRHV-1 and SbarRHV-1). This was carried out using virus-specific primer pairs and a diverse selection of blood and tissue samples. PCR-positive samples were not found, suggesting that these viruses are, at best, not common in the domestic pig populations tested (data not shown). This is in agreement with our recently published work, in which no herpesviruses other than pseudorabiesvirus, PCMV and PLHV-1, -2 and -3 were found in domestic pig populations (Chmielewicz et al., 2003b). However, based on data from this study, we cannot exclude the possible existence of a genogroup II gammaherpesvirus that naturally infects domestic pigs. Gammaherpesviruses of both genogroups are harboured by S. barbatus (PLHV-1, SbarLHV-1 and SbarRHV-1) and by B. babyrussa (PLHV-3 and BbabRHV-1). In domestic pigs, the distant relationship of a putative genogroup II gammaherpesvirus to the PLHVs may preclude its detection by PCR-based assays developed for PLHVs. Moreover, the virus would share much more identity with HHV-8 than with PLHVs. HHV-8 has been implicated in the pathogenesis of Kaposi's sarcoma, pri<1?show=[fo]>mary effusion lymphoma and Castleman's disease of plasma cell type (Schultz, 1998; Boshoff & Weiss, 2001). It has also been discussed as a potential pathogen in allotransplantation (Regamey et al., 1998). A related porcine virus might thus be a potential risk factor in xenotransplantation. Further investigation is currently under way to address this important safety concern.

In this study, evidence for the existence of beta- and gammaherpesviruses in Suidae (other than S. scrofa) has been reported for the first time. The observation for two groups of artiodactyl herpesviruses within the Gammaherpesvirinae sheds new light on the viral relationships in this herpesvirus subfamily and will contribute to further development in herpesvirus taxonomy.


   ACKNOWLEDGEMENTS
 
We thank Annette Kluge and Sigfried Pociuli for excellent technical assistance and Ursula Erikli for copy-editing the manuscript. We also thank Justin Seymour-Smith at Iwaba Wildlife Estate (Zimbabwe), James Burton, Alastair Macdonald, Cats Lyle, Michael Cantrell, Kristin Leus, Francis Vercammen, Wolfgang Dressen, Peter Bircher, Willem Schaftenaar, Marc de Boer, Kumar Pillai and Paolo Martelli (Singapore Zoo), Bapak Stany Subakir and staff (Surabaya Zoo), Andrew Kitchener (Royal Museums of Scotland) and The University of Edinburgh Development Trust.


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Received 12 November 2003; accepted 18 December 2003.