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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
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) and antibody-based (S. Brema, M. Goltz & B. Ehlers, unpublished) detection methods were therefore developed to allow monitoring of the PLHV status of donor pigs and xenotransplanted patients.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.
|
|
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 (156161 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
).
|
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Auchincloss, H., Jr & Sachs, D. H. (1998). Xenogeneic transplantation. Annu Rev Immunol 16, 433470.[CrossRef][Medline]
Boshoff, C. & Weiss, R. A. (2001). Epidemiology and pathogenesis of Kaposi's sarcoma-associated herpesvirus. Philos Trans R Soc Lond Ser B Biol Sci 356, 517534.[Medline]
Chmielewicz, B., Goltz, M. & Ehlers, B. (2001). Detection and multigenic characterization of a novel gammaherpesvirus in goats. Virus Res 75, 8794.[CrossRef][Medline]
Chmielewicz, B., Goltz, M., Franz, T. & 8 other authors (2003a). A novel porcine gammaherpesvirus. Virology 308, 317329.[CrossRef][Medline]
Chmielewicz, B., Goltz, M., Lahrmann, K. H. & Ehlers, B. (2003b). Approaching virus safety in xenotransplantation: a search for unrecognized herpesviruses in pigs. Xenotransplantation 10, 349356.[CrossRef][Medline]
Crawford, T. B., O'Toole, D. & Li, H. (1998). Malignant catarrhal fever. In Current Veterinary Therapy IV: Food Animal Practice, pp. 306309. Edited by J. L. Howard. Philadelphia: W. B. Saunders.
Eberle, R. & Hilliard, J. (1995). The simian herpesviruses. Infect Agents Dis 4, 5570.[Medline]
Ehlers, B., Borchers, K., Grund, C., Frölich, K., Ludwig, H. & Buhk, H.-J. (1999a). Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers. Virus Genes 18, 211220.[CrossRef][Medline]
Ehlers, B., Ulrich, S. & Goltz, M. (1999b). Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses. J Gen Virol 80, 971978.[Abstract]
Ehlers, B., Ochs, A., Leendertz, F., Goltz, M., Boesch, C. & Mätz-Rensing, K. (2003). Novel simian homologues of EpsteinBarr virus. J Virol 77, 1069510699.
Felsenstein, J. (1996). PHYLIP (phylogeny inference package) version 3.5. Department of Genetics, University of Washington, Seattle.
Ferry, J. A. & Harris, N. L. (1994). Lymphoproliferative disorders following organ transplantation. Adv Pathol Lab Med 7, 359387.
Fishman, J. A. (1994). Miniature swine as organ donors for man: strategies for prevention of xenotransplant-associated infections. Xenotransplantation 1, 4757.
Goltz, M., Widen, F., Banks, M., Belak, S. & Ehlers, B. (2000). Characterization of the DNA polymerase locus of porcine cytomegaloviruses from diverse geographic origins. Virus Genes 21, 249255.[CrossRef][Medline]
Goltz, M., Ericcson, T., Huang, C., Patience, C., Sachs, D. H. & Ehlers, B. (2002). Sequence analysis of the genome of porcine lymphotropic herpesvirus 1 and gene expression during post-transplant lymphoproliferative disease of pigs. Virology 294, 383393.[CrossRef][Medline]
Heuschele, W. P. (1988). Malignant catarrhal fever: a review of a serious disease hazard for exotic and domestic ruminants. Zool Garten NF 58, 123133.
Huang, C. A., Fuchimoto, Y., Gleit, Z. L. & 10 other authors (2001). Posttransplantation lymphoproliferative disease in miniature swine after allogeneic hematopoietic cell transplantation: similarity to human PTLD and association with a porcine gammaherpesvirus. Blood 97, 14671473.
O'Connell, P., Cunningham, A. & d'Appice, A. J. F. (2000). Xenotransplantation: its problems and potential as a clinical procedure. Transplant Rev 14, 1840.
Onions, D., Cooper, D. K. C., Alexander, T. J. L. & 9 other authors (2000). An approach to the control of disease transmission in pig-to-human xenotransplantation. Xenotransplantation 7, 143155.[CrossRef][Medline]
Regamey, N., Tamm, M., Wernli, M., Witschi, A., Thiel, G., Cathomas, G. & Erb, P. (1998). Transmission of human herpesvirus 8 infection from renal-transplant donors to recipients. N Engl J Med 339, 13581363.
Reid, H. W. & Buxton, D. (1989). Malignant catarrhal fever and the Gammaherpesvirinae of Bovidae. In Herpesvirus Diseases of Cattle, Horses and Pigs, pp. 116162. Edited by G. Wittmann. Boston: Kluwer.
Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. (2000). TREE-PUZZLE 5.0. Maximum likelihood analysis for nucleotide, amino acid, and two-state data.
Schultz, T. F. (1998). Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8). J Gen Virol 79, 15731591.
Tucker, A., Belcher, C., Moloo, B., Bell, J., Mazzulli, T., Humar, A., Hughes, A., McArdle, P. & Talbot, A. (2002). The production of transgenic pigs for potential use in clinical xenotransplantation: microbiological evaluation. Xenotransplantation 9, 191202.[CrossRef][Medline]
Ulrich, S., Goltz, M. & Ehlers, B. (1999). Characterization of the DNA polymerase loci of the novel porcine lymphotropic herpesviruses 1 and 2 in domestic and feral pigs. J Gen Virol 80, 31993205.
van Zanten, J., de Leij, L., Prop, J., Harmsen, M. C. & The, T. H. (1998). Human cytomegalovirus: a viral complication in transplantation. Clin Transplant 12, 145158.[Medline]
Zimmermann, W., Broll, H., Ehlers, B., Buhk, H.-J., Rosenthal, A. & Goltz, M. (2001). Genome sequence of bovine herpesvirus 4, a bovine rhadinovirus, and identification of an origin of DNA replication. J Virol 75, 11861194.
Received 12 November 2003;
accepted 18 December 2003.