1 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
2 University of Stirling, UK
3 Institute of Zoology, London, UK
4 University of Edinburgh, UK
Correspondence
Colin McInnes
mcinc{at}mri.sari.ac.uk
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ABSTRACT |
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Present address: University of Otago, Dunedin, New Zealand.
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MAIN TEXT |
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Experimental infection with a mixture of isolates from various outbreaks of scab disease confirmed that SPPV is the agent responsible for the disease observed in red squirrels. In contrast, infection of grey squirrels failed to induce disease although all developed antibodies to the virus (Tompkins et al., 2002). We have taken a single isolate of the virus from an outbreak of disease in northern England and shown that it alone causes a deleterious disease in red squirrels. Sequencing and subsequent phylogenetic analysis of genes from this virus casts doubt over its relationship to the parapoxviruses.
Virus was isolated from a red squirrel (see supplementary data at JGV Online: http://vir.sgmjournals.org) with typical SPPV-induced haemorrhagic erythematous dermatitis about the face and on the feet and was visualized by negative-staining transmission electron microscopy. The SPPV particles were generally ovoid with approximate dimensions of 275x175 nm, similar to the parapoxviruses. The regular basket-weave surface morphology characteristic of the parapoxviruses was also observed (Fig. 1A). However, the angle at which the SPPV basket-weave ridges cross each other appears to differ from that of other parapoxviruses, supporting the previous observations made by Scott et al. (1981)
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SPPV has been formally classified as a Parapoxvirus (van Regenmortel et al., 2000), although Sands et al. (1984)
concluded that SPPV and Orf virus (ORFV) were antigenically distinct. Furthermore, in a study of 27 monoclonal antibodies (mAbs) raised against ORFV, only two were found to cross-react with SPPV (Housawi et al., 1998
). This contrasts with 17 mAbs that cross-reacted with both Pseudocowpox virus (PCPV) and Bovine papular stomatitis virus (BPSV) and six that cross-reacted with Sealpox virus, suggesting that SPPV is more divergent from ORFV than from these other parapoxviruses. To study the relationship between SPPV and the other parapoxviruses we attempted to identify genes within the SPPV genome that were related to known ORFV genes and in particular to those associated with modulation of the host immune response and/or virulence. Five cosmid clones that span the SPPV genome were screened for the presence of sequence related to the ORFV GIF gene (encoding a protein capable of binding interleukin-2 and granulocytemacrophage colony-stimulating factor; Deane et al., 2000
), the VEGF-E gene (encoding a protein related to mammalian vascular endothelial growth factor; Lyttle et al., 1994
), the IL-10 gene (encoding a protein closely related to mammalian interleukin-10; Fleming et al., 1997
) and the orthologue of the H5R gene of Vaccinia virus (VACV) (encoding a late transcription factor, VLTF-4; Kovacs & Moss, 1996
). Two of these genes, VEGF-E and GIF, have been found so far only in parapoxviruses whilst the IL-10 gene has also been found in the capripoxvirus Lumpy skin disease virus (LSDV) (Tulman et al., 2001
). Orthologues of H5R are found in each of the chordopoxvirus genera. No specific hybridization between the ORFV probes and SPPV DNA was detected with the exception of that corresponding to the H5R gene (results not shown). On the basis of this it was hypothesized that SPPV does not encode orthologues of any of the ORFV GIF, VEGF or IL-10 genes or that the corresponding SPPV sequences have diverged sufficiently so as to prevent cross-hybridization. Comparative sequence analysis of 22 ORFV strains and isolates demonstrated that the VEGF-E gene varies considerably even within a single species of virus (Mercer et al., 2002
). This heterogeneity could explain our inability to identify an orthologue of VEGF-E in SPPV by hybridization. VEGF-E genes are present close to the right ITR junctions in two of the three parapoxviruses for which there is sequence data available, that is in ORFV and PCPV, but not in BPSV (Lyttle et al., 1994
; Rziha et al., 2003
; Ueda et al., 2003
). We sequenced approximately 1 kb of the corresponding region of the SPPV genome, but were unable to identify an orthologue of VEGF-E. Instead an ORF with similarity to the 003L and 157R genes of Molluscum contagiosum virus (MOCV) was found (results not shown). These genes appear to represent paralogues of each other although they do differ at their 3' ends (Senkevich et al., 1997
). The SPPV ORF (GenBank accession no. AY312570) is approximately 49 % identical to the MOCV sequences, with the predicted amino acid sequences sharing approximately 28 % identity.
The hybridization and sequence data taken together suggest that SPPV probably does not possess an orthologue of ORFV VEGF-E. This was unexpected because the appearance of SPPV-induced lesions suggests an underlying vascularization similar to that of ORFV-induced lesions. The vascularization appears to be a function of VEGF-E since knockout viruses lacking this protein produced lesions that had lower blood vessel formation in the dermis than lesions induced with wild-type virus and as a consequence were considerably less erythematous and florid (Savory et al., 2000).
Since we were unable to identify known parapoxvirus genes associated with immunomodulation or virulence within the SPPV genome we sought alternative genes with which to explore the relationship between SPPV and other parapoxviruses. Phylogenetic analysis with the partial sequence of the major outer envelope protein (orthologue of VACV F13L) has been used to infer speciation within the genus Parapoxvirus (Becher et al., 2002). Previous analysis was based on the alignment of amino acids 137 to 320 (numbering with respect to the ORFV protein) because the data produced for the majority of parapoxviruses are derived from PCR products using primers considered to be specific for all parapoxviruses (Inoshima et al., 2000
). Attempts to obtain a PCR product from SPPV DNA using these primers failed. Nevertheless we identified, by DNA hybridization with the ORFV gene, a plasmid containing the corresponding SPPV sequence. Sequence analysis revealed the presence of an ORF (GenBank accession no. AY312569) corresponding to amino acids 129 to 344 of the ORFV protein. Pairwise alignment of the predicted SPPV amino acid sequence with that of the four recognized species of Parapoxvirus, ORFV, PCPV, BPSV and Parapoxvirus of red deer in New Zealand (PVNZ), and the tentative member of the genus, Sealpox virus, indicated that the SPPV protein shared approximately 49 % identity to each. This was less than with VACV F13L (57 % identity). We aligned our sequence with all known chordopoxvirus orthologues of the F13L protein, representing all eight chordopoxvirus genera, and constructed a phylogenetic tree based on a maximum-likelihood algorithm (Fig. 2
A). The analysis grouped ORFV, BPSV, PCPV and PVNZ together with Sealpox virus, but the SPPV sequence was placed on a separate branch of the tree with 82 % support from the bootstrap resampling. Indeed, the SPPV sequence was placed on a branch of its own and did not partition with any of the other poxvirus genera, suggesting that SPPV represents a separate genus of the Poxviridae.
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Despite similarities in the base composition, virion morphology and in the pathological symptoms of the disease caused by SPPV when compared to the other parapoxviruses, the phylogenetic analysis presented here provides no support to classify SPPV in the genus Parapoxvirus. However, a much more extensive study of the SPPV genome and the genes it contains will be required before the virus can be classified with certainty. This classification will be important when considering future epidemiological and ecological studies.
Whilst preparing this manuscript we noted that the standard abbreviation given to the Squirrelpox virus (SPPV) is the same as has been given to Sheeppox virus (van Regenmortel et al., 2000). We would like to suggest that the alternative abbreviation SQPV be adopted for Squirrelpox virus.
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ACKNOWLEDGEMENTS |
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Received 26 June 2003;
accepted 1 September 2003.