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Short Communication |
1 National Veterinary and Food Research Institute, Department of Virology, PO Box 45, Hämeentie 57, FIN-00581 Helsinki, Finland
2 Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 OPZ, UK
3 Virus Research Unit, Department of Microbiology, University of Otago, Dunedin, New Zealand
4 Federal Research Centre for Virus Diseases of Animals, Institute of Immunology, Paul-Ehrlich-Strasse 28, D-72076 Tübingen, Germany
5 CSC – Scientific Computing Ltd, PO Box 405, FIN-02101 Espoo, Finland
6 National Veterinary and Food Research Institute, Oulu Regional Unit, PO Box 517, FIN-90101 Oulu, Finland
Correspondence
Maria K. Tikkanen
maria.tikkanen{at}eela.fi
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this article are AY453652–AY453689 and AY455291–AY455311.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), demonstrated that the outbreak was caused by a parapoxvirus (PPV). PPVs, members of the Poxviridae, are highly contagious, being transmitted by direct contact between animals or indirectly via environmental contamination (Haig & Mercer, 1998). Worldwide they are the cause of contagious pustular dermatitis in sheep and goats (Orf virus, ORFV) and in cattle (Pseudocowpoxvirus, PCPV, and Bovine papular stomatitis virus, BPSV; Moyer et al., 2000), while a less severe disease is found in Red deer in New Zealand (Parapoxvirus of red deer in New Zealand, PVNZ).
The aetiological agent of the disease in reindeer is not known, although based on clinical symptoms and pathology, ORFV was thought to have been the main cause of the 1992–1993 outbreak. Here we report the findings of an investigation into the cause of an outbreak of disease in 1999–2000, and compare the results with those obtained from the earlier outbreak. PCR was used to amplify specific regions of PPV genomes direct from clinical specimens, and phylogenetic analysis of the resulting PCR products was used to determine the most likely cause of disease. Since Rangiferine herpesvirus 1 (RanHV-1) has been shown to be prevalent in Finnish reindeer (Ek-Kommonen et al., 1982, 1986), and a bovine herpesvirus 1-like virus has been isolated from clinically similar cases of disease in reindeer in Sweden (Rockborn et al., 1990), the samples were also analysed with PCR specific for these ruminant alphaherpesviruses (Ros & Belák, 1999).
Eighty-one clinical samples, from 57 reindeer showing symptoms of papular stomatitis during the winter of 1999–2000, were collected for PCR analysis. The samples, both scabs and vesicle swabs, originated from different parts of northern Finland. DNA was purified from the samples using the QIAamp DNA Mini kit (Qiagen). Similarly, DNA was obtained from several PPV reference strains (Fig. 1b) grown in vitro. DNA was also extracted from a further four clinical specimens: two reindeer scab samples taken in northern Finland in 1992 and 1994; a paraffin wax-embedded tissue block obtained from clinically ill sheep in 1997 from northern Finland; and a paraffin-block sample taken from a cow during a severe outbreak in Parainen (south-west Finland) in 1999. The DNA was purified from paraffin blocks essentially as described by Jackson et al. (1990). Finally, two PPV DNA samples from Germany (PPV strain BO29 and PCPV strain BO35) were included in the studies. BO29 was isolated from a person who had had close contact with sheep and had suffered typical orf lesions (isolate BO15; Büttner & Rziha, 2002), and BO35 is a PPV strain isolated in 2000 from a cow's teat in Germany.
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; Goebel et al., 1990). The ORFV-specific primer set F1 (5'-TCA ATA TGG ATG AAA ATG AC-3') and F2 (5'-ACA GAC GGC AAC ACA GCG GT-3') was designed to amplify a 301 bp product of the ORFV orthologue of the VACV fusion protein gene A27L (Rodriguez & Esteban, 1987; Goebel et al., 1990). After initial denaturation at 99 °C for 8 min, AmpliTaq polymerase (Perkin Elmer) was added during an 80 °C incubation period of 10 min, and the amplifications were performed for 35 cycles of denaturation (95 °C, 1 min), annealing (58 °C, 1 min for the C1/C2 and C3/C4 primers; 47 °C, 1 min for the F1/F2 primers) and extension (72 °C, 1 min). The RanHV-1-specific PCR was performed as described by Ros & Belák (1999). Positive samples from the PPV-specific PCRs were subjected to the semi-nested PCR described by Inoshima et al. (2000), which is designed to amplify 594 and 235 bp products of the PPV orthologue of the VACV gene F13L encoding the major envelope antigen p37K.
Amplification products were obtained from all the PPV reference strains with at least one of the PPV-specific primer pairs. Samples from ten reindeer (18 %) from the 1999–2000 outbreak were found to be positive in the PPV-specific core protein PCRs, with the remaining being negative in all the PCR assays. Each of the ten positives was also positive in the semi-nested PCR (Inoshima et al., 2000), although only three gave amplicons of 594 bp after the first round of PCR. None of the ten positive reindeer samples was positive in either the ORFV- or RanHV-1-specific PCRs, suggesting that at least a proportion of the papular stomatis observed in the 1999–2000 outbreak in Finnish reindeer could be attributed to a PPV other than ORFV. The results demonstrated that the conserved core protein, in particular, is an acceptable target for diagnostic PCR since the combination of primers used detects all recognized PPVs.
To characterize the viral DNA isolated from these reindeer in more detail, the PCR products were sequenced and subjected to phylogenetic analyses. Sequencing was performed at the DNA Synthesis and Sequencing Laboratory, Institute of Biotechnology, University of Helsinki. The sequences of both strands of the PCR products were determined using PCR primers, although the primer sequences were subsequently excluded from the analyses. The conceptual amino acid sequences of each of the PCR products were obtained using the program Transeq of the EMBOSS software package version 2.6.0.
Pairwise comparisons of both DNA and amino acid sequences corresponding to both regions of the major core protein and the major envelope protein revealed that reindeer PPV isolates from 1999–2000 showed 96–100 % nucleotide and amino acid identity with PCPV strains BO35 and VR634 and an isolate from a Finnish cow (F99.177C), whereas the reindeer PPV isolates from earlier years (F92.849R and F94.848R) showed the highest nucleotide and amino acid identity with ORFV reference strains. Fig. 1(a)
shows the alignment of the predicted envelope protein sequences of PPVs displaying the most variable sites among the protein regions studied.
To analyse further the genetic relationships between Finnish PPVs, reference PPVs and other members of the Chordopoxvirinae, phylogenetic trees were constructed from alignments of the conceptual core and envelope protein sequences. These analyses were performed with the PHYLIP package version 3.6b (Felsenstein, 2003). Phylogenetic trees were inferred using distance, parsimony and maximum-likelihood methods. The model of amino acid substitution chosen for both distance and maximum-likelihood methods was the Jones–Taylor–Thornton model (Jones et al., 1992) with four gamma rates. The gamma distribution parameter alpha and the relative rate for gamma rate categories were calculated using the program TREE-PUZZLE version 5.1 (Schmidt et al., 2002). The phylogenetic trees were inferred from the distance matrices using the neighbour-joining method. Maximum parsimony analyses were carried out using the program PROTPARS, and maximum-likelihood analyses (with randomized input order and global rearrangements) were performed using the program PROML. The reliability of the trees was determined by 1000 data-set bootstrap resampling with the programs SEQBOOT and CONSENSE.
Because the trees did not differ significantly, only the maximum-likelihood trees are presented (Fig. 2a–c). The results show that PPVs form three or four phylogenetic lineages depending on the virus species included in the analyses. The lineages are in accordance with the established PPV genera. PPVs were always clearly separated from other chordopoxviruses (ChPVs), which is consistent with the fact that PPVs differ from the other ChPVs in their morphology and mostly also in their genome size (Moyer et al., 2000). The results obtained with the different methods were consistent in that ChPVs were always seen in five main groupings: Parapoxvirus; Orthopoxvirus; Capripovirus/Suipoxvirus/Leporipoxvirus/Yatapoxvirus; Avipoxvirus; and Molluscipoxvirus. Trees calculated from all gene regions with all methods revealed that F99/00.R strains were clustered in the same lineage with the PCPV strains VR634 and BO35 and the isolate from a cow (F99.177C), while F92.R, F92.849R and F94.848R were grouped with the ORFV strains and an isolate from a sheep (F97.391S), all with high bootstrap support.
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). Most PPV samples with missing data were excluded from the analysis in order to improve the likelihood of finding a fully resolved tree topology. The tree obtained from concatenated data is presented in Fig. 3. As expected, reindeer PPV strains from 1992–1994 grouped together with ORFV, and the reindeer PPV strains from the recent outbreak grouped with PCPV, with high bootstrap support. The tree also verifies the results obtained from the single gene analyses, displaying the same five main groupings of ChPVs. These results are in accordance with recent data published by Gubser et al. (2004): their results from the combined phylogenetic analysis of 17 conserved proteins from all ChPV genera except the PPVs showed the same main grouping of ChPVs, minus the PPPVs, that we found in our study.
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), which suggests there is little variation between PPVs originating from different animal species and from different geographical areas. However, further analysis of the reindeer PPV 1999–2000 isolates will be required before it can be determined for certain whether the limited differences found between reindeer PPV and virus isolated from a cow (F99.177C) represent adaptation of the same virus to different host species, or indicate separate virus species. Transmission of a PPV from cow to reindeer, or vice versa, cannot be excluded at this time even though there are no obvious connections between the various outbreaks of disease in reindeer and in cattle. Indeed, PPV infection in cattle is not considered a serious problem in Finland, despite the recent epidemic in Parainen in 1999 and two previous outbreaks, one in south-west Finland in 1971 (Estola & Neuvonen, 1974) and the other in Häme in 1974 (Kuokkanen & Launis, 1975), all of which were geographically remote from where the affected reindeer were found. Recently, Tryland et al. (2001) also showed that infection of semi-domesticated reindeer in Norway was caused by PPV. Further characterization of the Norwegian virus will show whether the symptoms in Finland and Norway are caused by the same virus. This is the first time sequence analysis has shown that the outbreaks of papular stomatis in Finnish reindeer may result from infection with different species of parapoxvirus. Further work is required to determine whether the outbreaks of disease in 1992–1994 and 1999–2000 were due to infection with ORFV and PCPV, respectively, or whether the disease is caused by a separate virus species closely related to both ORFV and PCPV. If reindeer are susceptible to both ORFV and PCPV this will have implications for animal husbandry in terms of taking care to minimize contact between reindeer and potential sources of infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Büttner, M., von Einem, C., McInnes, C. J. & Oksanen, A. (1995). Klinik und Diagnostik einer schweren Parapocken-Epidemie beim Rentier in Finnland. Tierärztl Prax 23, 614–618 (abstract in English).[Medline]
Ek-Kommonen, C., Veijalainen, P., Rantala, M. & Neuvonen, E. (1982). Neutralizing antibodies to bovine herpesvirus 1 in reindeer. Acta Vet Scand 23, 565–569.[Medline]
Ek-Kommonen, C., Pelkonen, S. & Nettleton, P. (1986). Isolation of a herpesvirus serologically related to bovine herpesvirus 1 from a reindeer (Rangifer tarandus). Acta Vet Scand 27, 299–301.[Medline]
Estola, T. & Neuvonen, E. (1974). Valelehmärokkovirus vedinrokon aiheuttajana. Pseudocowpox virus as a cause of teat pox in Finland. Suomen Eläinlääkärilehti 80, 622–629 (abstract in English).
Felsenstein, J. (2003). PHYLIP (Phylogeny Inference Package) version 3.6b. Distributed by the author. Seattle, WA, USA: Department of Genetics, University of Washington.
Gassmann, U., Wyler, R. & Wittek, R. (1985). Analysis of parapoxvirus genomes. Arch Virol 83, 17–31.[CrossRef][Medline]
Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P. & Paoletti, E. (1990). The complete DNA sequence of vaccinia virus. Virology 179, 247–266.[CrossRef][Medline]
Gubser, C., Húe, S., Kellam, P. & Smith, G. L. (2004). Poxvirus genomes: a phylogenetic analysis. J Gen Virol 85, 105–117.
Haig, D. M. & Mercer, A. A. (1998). Orf. Vet Res 29, 311–326.[Medline]
Huelsenbeck, J. P., Bull, J. J. & Cunningham, C. W. (1996). Combining data in phylogenetic analysis. TREE 11, 152–158.
Inoshima, Y., Morooka, A. & Sentsui, H. (2000). Detection and diagnosis of parapoxvirus by the polymerase chain reaction. J Virol Methods 84, 201–208.[CrossRef][Medline]
Jackson, D. P., Lewis, F. A., Taylor, G. R., Boylston, A. W. & Quirke, P. (1990). Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. J Clin Pathol 43, 499–504.
Jones, D. T., Taylor, W. R. & Thornton, J. M. (1992). The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8, 275–282.
Kuokkanen, K. & Launis, J. (1975). Lypsäjänkyhmyepidemia pirkanmaalla syksyllä 1974. An epidemic of milker's nodules in Finland. Duodecim 91, 769–774 (abstract in English).[Medline]
Mayr, A., Herlyn, M., Mahnel, H., Zach, A. & Bosted, H. (1981). Bekämpfung des Ecthyma contagiosum (Pustulardermatitis) der Schafe mit einem neuen Parenteral-Zellkultur-Lebendimpfstoff. J Vet Med B 28, 535–552.
McInnes, C. J., Wood, A. R., Nettleton, P. F. & Gilray, J. A. (2001). Genomic comparison of an avirulent strain of Orf virus with that of a virulent wild type isolate reveals that the Orf virus G2L gene is non-essential for replication. Virus Genes 22, 141–150.[CrossRef][Medline]
Moyer, R. W., Arif, B. M., Black, D. N. and ten others (2000). Family Poxviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 137–157. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Page, R. D. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Robinson, A. J. & Mercer, A. A. (1995). Parapoxvirus of red deer: evidence for its inclusion as a new member in the genus Parapoxvirus. Virology 208, 812–815.[CrossRef][Medline]
Robinson, A. J., Ellis, G. & Balassu, T. C. (1982). The genome of orf virus: restriction endonuclease analysis of viral DNA isolated from lesions of orf in sheep. Arch Virol 71, 43–55.[CrossRef][Medline]
Rockborn, G., Rehbinder, C., Klingeborn, B., Leffler, M., Klintevall, K., Nikkilä, T., Landen, A. & Nordkvist, M. (1990). The demonstration of a herpesvirus, related to bovine herpesvirus 1 in reindeer with ulcerative and necrotizing lesions of the upper alimentary tract and nose. Rangifer Special Issue 3, 373–384.
Rodriguez, J. F. & Esteban, M. (1987). Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein. J Virol 61, 3550–3554.
Ros, C. & Belák, S. (1999). Studies of genetic relationships between bovine, caprine, cervine and rangiferine alphaherpesviruses and improved molecular methods for virus detection and identification. J Clin Microbiol 37, 1247–1253.
Rosel, J. & Moss, B. (1985). Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b. J Virol 56, 830–838.
Scagliarini, A., Ciulli, S., Battilani, M., Jacoboni, I., Montesi, F., Casadio, R. & Prosperi, S. (2002). Characterisation of immunodominant protein encoded by the F1L gene of orf virus strains isolated in Italy. Arch Virol 147, 1989–1995.[Medline]
Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. (2002). TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, 502–504.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882.
Tryland, M., Josefsen, T. D., Oksanen, A. & Aschfalk, A. (2001). Parapoxvirus infection in Norwegian semi-domesticated reindeer (Rangifer tarandus tarandus). Vet Rec 149, 394–395.
Received 5 November 2003;
accepted 9 February 2004.
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