Squirrelpox virus (SQPV) shows little evidence for morbidity or mortality in North American grey squirrels (Sciurus carolinensis), in which the virus is endemic. However, more recently the virus has emerged to cause epidemics with high mortality in Eurasian red squirrels (S. vulgaris) in Great Britain, which are now threatened. Here we report the genome sequence of SQPV. Comparison with other Poxviridae revealed a core set of poxvirus genes, the phylogeny of which showed SQPV to be in a new Chordopoxvirus subfamily between the Molluscipoxviruses and Parapoxviruses. A number of SQPV genes were related to virulence, including three major histocomaptibility class I homologs, and one CD47 homolog. In addition, a novel potential virulence factor showing homology to mammalian oligoadenylate synthetase (OAS) was identified. This family of proteins normally causes activation of an endoribonuclease (RNaseL) within infected cells. The putative function of this novel SQPV protein was predicted in silico.
Citation: Darby AC, McInnes CJ, Kjær KH, Wood AR, Hughes M, Martensen PM, et al. (2014) Novel Host-Related Virulence Factors Are Encoded by Squirrelpox Virus, the Main Causative Agent of Epidemic Disease in Red Squirrels in the UK. PLoS ONE 9(7): e96439. https://doi.org/10.1371/journal.pone.0096439
Editor: Xiangzhi Meng, University of Texas HSC at San Antonio, United States of America
Received: January 10, 2013; Accepted: April 8, 2014; Published: July 1, 2014
Copyright: © 2014 Darby et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: We thank the Wildlife Ark Trust for their funding of this work, as part of their overall strategy to develop a vaccine against squirrel poxvirus. ACD was funded by the Royal College of Veterinary Surgeons trust. KHK and PMM were supported by the The Danish Council for Independent Research/Natural Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Poxviruses cause disease in many different taxa and have been described in insects, birds, reptiles and mammals . The mammalian poxviruses are found both in terrestrial and marine environments with many restricted to one host species, but a number can cause disease in more than one species with some being zoonotic . Squirrelpox virus (SQPV) has emerged in Great Britain as the cause of high mortality in Eurasian red squirrel (Sciurus vulgaris) populations whilst apparently leaving sympatric North American grey squirrel (Sciurus carolinensis) populations unaffected. The underlying mechanisms responsible for the pathogenicity of SQPV are not understood and hence the reason for the different disease manifestation in the two species cannot be fully investigated. In this paper we document the complete sequence of the SQPV genome (excluding the inverted terminal repeats), confirming that it is a species in the family Poxviridae and does not partition with any of the currently recognized genera.
The disease and epidemiology
Red squirrels with squirrelpox show typical signs associated with poxvirus infections, with the formation of multifocal extensive ulcerative lesions around the mouth and eyelids . Squirrelpox lesions contain high concentrations of virus which can be shed to the environment, possibly helping to sustain infection. Diseased individuals rarely survive infection and this has had a profoundly negative impact on the distribution of red squirrels in Great Britain , , . Red squirrels only become infected where they are sympatric with the introduced North American grey squirrels which are considered to be the reservoir host of the virus. Although most grey squirrels in England and Wales appear to be serologically positive for antibodies to the virus, they rarely show signs of disease and indeed currently there are no known negative consequences of SQPV infection in grey squirrels , .
It had been postulated that competition for habitat and resources partially explains the replacement of reds by greys , however epidemiological modelling of squirrelpox suggests that grey squirrels replace red squirrel populations up to 25 times faster when the grey squirrel populations carry the poxvirus infection . Current estimates put the remaining UK population of red squirrels at approximately 160,000 with 75% of those being in Scotland, predominantly in areas where there are either no grey squirrels or where reds are sympatric with grey squirrels which are serologically negative for SQPV. It has been suggested that the red squirrels will die out within 15–20 years if nothing is done to protect them from SQPV.
Virulence genes and mechanisms
Viruses often encode proteins related to the immune system of their host in order to circumvent host immunity and improve viral survival. Poxvirus-encoded virulence factors target many different aspects of host immunity. The majority act to block or subvert the host anti-viral responses, and include proteins that manipulate apoptosis of host cells, inhibit the activity of both natural killer (NK) cells and cytotoxic T-cells and that bind and/or inhibit a multitude of host cytokines , , , , , .
The interferon (IFN) response system is targeted in many ways by poxvirus-encoded factors acting before and after IFN production . Usually this involves binding and neutralization of IFN molecules [e.g. some poxviruses encode soluble IFN receptor homologues which prevent IFN action) or other proteins functioning in the IFN system . Poxviruses also target specific enzymes in IFN-dependent pathways, such as protein kinase R [PKR). By targeting these enzymes the poxviruses help to prevent the shutdown of viral protein synthesis in the infected cell . Outside of the poxvirus family there are other viruses that encode transcription suppressors of the IFN system (the HHV-8 virus encodes an IRF homolog which suppresses transcription) and viruses that encode RNA binding proteins preventing activation of PKR and oligoadenylate synthetase OAS)/RNaseL .
The emergence of SQPV in red squirrels raises important questions about the virus's evolution and it's host interactions. It is clear SQPV has the ability to transfer between host species and has coevolved mechanisms which allow attenuated pathogenesis and virulence within the grey squirrel resulting in asymptomatic infection, but producing a high degree of virulence in the red squirrel host.
Here we describe the genomic sequence of SQPV, discuss its phylogenetic relationship with the other Poxviridae, and describe novel virulence genes including one predicted to impede the host interferon response via interaction with the OAS system.
Materials and Methods
The virus isolate used in this study was a from a necropsy sample taken from a red squirrel that had been found dead in the wild as previously described in . The work was therefor not subject to animal welfare legislation in the UK and ethical permission for sampling was not sought.
DNA Cloning and sequencing
Pox genomic DNA isolated directly from scabs from an infected squirrel  was cloned into SuperCos I cosmids (Stratgene, La Jolla, USA) . Six cosmids with an average insert size of 35 kb were selected on the basis that they spanned the entire genome. DNA was prepared using the Plasmid Maxi Kit (Qiagen, UK) and pooled in equimolar proportions before being made into a single fragment shotgun sequencing library. Sequencing was conducted on a Genome Sequencer FLX System (Roche Diagnostics, UK) and assembled using Newbler (Roche Diagnostics, UK).
Assembly finishing and annotation
After assembly the resulting contigs were filtered with BLAST to remove the non-viral sequences. The contigs were viewed in GAP4 (http://staden.sourceforge.net) and gaps closed by assembling Sanger sequences. Protein-coding genes were identified by GLIMMER , GenemarkS eukaryotic viruses model  and BLASTX using a viral protein database downloaded from NCBI FTP site (August 2009). Putative functions were inferred using BLAST against the NCBI databases and InterProScan . Artemis v11 was used to organize data and facilitate annotation . A full list of annotated genes is in Table S1.
The pox phylogeny was reconstructed using orthologous gene sets identified in other viral genomes using ORTHOMCL (default parameters), 39 orthologous gene clusters where identified across 19 pox taxa (Canarypox virus CNPV NC_005309; Fowlpox virus FWPV NC_002188; Sheeppox virus 17077-99 SPPV NC_004002; Goatpox virus Pellor GTPV NC_004003; Lumpy skin disease virus NI-2490 LSDV NC_003027; Rabbit fibroma virus RFV NC_001266; Myxoma virus MYXV NC_001132; Molluscum contagiosum virus MOCV NC_001731; Monkeypox virus Zaire-96-I-16g MPXV NC_003310; Camelpox virus CMLV NC_003391; Ectromelia virus ECTV NC_004105; Vaccinia virus VACV NC_006998; Cowpox virus CPXV NC_003663; Orf virus ORFV NC_005336; Bovine papular stomatitis virus BPSV NC_005337; Swinepox virus SWPV NC_003389), aligned with MUSCLE  and trimmed with GBLOCKS  Alignments of 40 genes (orthologous genes to SQPV013c, SQPV022, SQPV024c, SQPV026c, SQPV029, SQPV030, SQPV036c, SQPV038c, SQPV040c, SQPV041c, SQPV044c, SQPV046, SQPV048, SQPV049, SQPV050, SQPV052, SQPV055c, SQPV056, SQPV058, SQPV060, SQPV065c, SQPV067c, SQPV073c, SQPV075, SQPV080, SQPV088c, SQPV090c, SQPV092c, SQPV095, SQPV096c, SQPV099, SQPV100c, SQPV101c, SQPV104c, SQPV110c, SQPV113c, SQPV114, SQPV115, SQPV116, SQPV122c) were then concatenated and maximum likelihood trees calculated by JTT, estimated transition/transversion ratio, fix proportion of invariable sites were implemented using PHYML , 1000 boot replicates were performed.
Structural homologues were identified using predicted amino acid sequences of relevant ORFs submitted for analysis on the SWISS-MODEL database . Estimated model reliability was calculated using QMEAN4 score (range 0–1, higher values indicate better fit). These data were displayed and manipulated in PYMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC).
Results and Discussion
The Poxviridae are dsDNA viruses with linear genomes that range in size from ca. 135 kb to 360 kb. The final assembly for the SQPV genome (containing only one of the inverted terminal repeat [ITR] sequences) was within this range at 148 kb (one contig) with a GC content of 66.69% (Table 1). The previously available genomic data for SQPV  covered 60 kb from the right and left ends of the genome (∼40% genome), but lacked the central region of the sequence thought mainly to contain genes conserved across all poxvirus genera. The partial sequence of one of the two inverted terminal (ITR) sequences at the left hand end is included in the final assembly. The right hand ITR has the same sequence and size as the left hand ITR (3383 bp), but could not be resolved separately due to its identical sequence. There is one gene (SQPV_001) predicted to span the ITR/unique sequence boundary at the left end of the genome, but none at the right end of the genome, justifying the inclusion of only one copy of the ITR in the final assembly. The genome is therefore complete for all protein coding regions in the unique region, but lacks predicted extreme left and right hand ends of the genome, including the covalently closed terminal loops. It was estimated previously that the ITR was approximately 5 kb in length  and therefore the full genome size is predicted to be between 152–155 kb. The genome sequence has been submitted to the National Center for Biotechnology Information (NCBI) databases under the accession HE601899.
Phylogentic position of squirrelpox virus
The phylogenetic analysis of the virus (Figure 1A) shows that squirrelpox virus is a member of the subfamily of chordopoxviruses. The tree topology and branch lengths suggest that SQPV shares a common ancestor with Molluscipoxvirus and the Parapoxviruses, but represents a distinct and novel genus. This was suggested previously  but this study provides a more robust phylogeny and is supported by multiple methods and individual gene trees (data not shown). The Phylogeny suggests a relatively close relationship with the Parapoxvirinae and the Molluscipoxvirinae, but the presence of the VACV-Cop F15 and D9 homologs in the SQPV genome, together with the positional conservation of the VACV-Cop F9 and F10 genes (cf. the parapoxviruses), differentiates SQPV from the parapoxviruses suggesting the viruses must have diverged before the gene rearrangements and losses occurred in the Parapoxvirinae.
A. Poxvirus phylogeny showing the novel position of the SQPV. Midpoint rooted ML tree based on 39 conserved core genes from 19 taxa. Node values give percentage bootstrap support (n = 1000). See Table 2 for abbreviations. B. SQPV genome overview showing conserved poxvirus genes in grey and unique SQPV genes in red (displayed according to their coding strand). The grey bands show orthologous genes found in other poxviruses (displayed without coding strand information).
Chordopoxvirus genomes have a central core of conserved genes. This consists of a minimum (core) set of 88 genes which contains genes essential for virus viability and encode proteins involved in viral DNA replication, transcription and virion assembly . These are all present in the SQPV genome (See Table S1) which also includes the extra two genes (VACV-Cop F15 and D9) that are reported to be found in all other chordopoxvirus genomes except the parapoxviruses . In the central conserved core region of the SQPV genome there are an additional 15 genes, 10 of which appear to be unique to SQPV. These 10 genes include two MHC class I-like molecules, an orthologue of 2′,5′-oligoadenylate synthetase (see below) and a likely member of the viral A-type inclusion protein superfamily. The remaining six have no known or predicted function. Eighty-two percent [115/141) of the genes in the genome have homologs in another pox genome, (Table S1). Most of the conserved genes are located in the middle of the genome (Figure 1B), whilst the more variable and novel genes that are likely to define host range and virulence (many of which have been shown by others to be non-essential for in vitro replication), are located at either end of the genome. Our data corrects mistaken annotation in the original description of these flanking regions .
Unlike the parapoxviruses and the molluscum contagiosum virus, SQPV contains a thymidine kinase (TK) gene in the central region of the genome similar to the majority of other poxviruses. The SQPV TK gene, however, is not positionally conserved, in comparison to other poxviruses, and BLAST database searching suggests its closest orthologue is from the rat (Rattus norvegicus) and other rodents. This might suggest that the SQPV TK gene is a relatively recent, independent, acquisition and further analysis of its sequence may help in determining some of the evolutionary history of the virus.
Putative immune modifiers
Major histocompatability complex (MHC) Class I.
Many viruses have evolved ways to interfere with natural killer (NK) cell activation  through encoding MHC Class I homologs. This allows the virus to 1) down-regulate host MHC expression and 2) avoid NK surveillance through surface expression of viral MHC homologs. Such viral MHC proteins are often encoded in herpesviruses, but rare in poxvirus genomes. The SQPV genome is unusual amongst poxviruses, encoding three putative MHC-like homologs (SQPV_004, SQPV_027 and SQPV_064), spread throughout the genome. SQPV_004 is most conserved with class I (127/291(44%) identity to human MHC class 1, AAA57146), whilst SQPV_027 and SQPV_064 are much less conserved (72/273 (26%) and 47/194 (24%) respectively) (Figure S1). The three genes share little identity to each other (SQPV_004 to SQPV_027 – 24%: 65/271 aa. SQPV_004 to SQPV_064 – 20%: 11/55 aa. SQPV_027 to SQPV_064 – 22%: 15/68 aa), which may suggest they represent independent acquisition events or arose as the result of an ancient duplication before subsequent divergence. Both SQPV_004 and SQPV_027 are predicted to have similar transmembrane domains to class I molecules, but SQPV_064 appears to lack a C terminal hydrophobic domain, suggesting it could be secreted. The poxvirus Molluscum contagiosum virus (MOCV) encodes one MHC class I homolog (MOCV080R gene product) . This protein is largely sequestered in the endoplasmic reticulum and Golgi, is capable of binding β2-microglobulin and is not detected on the cell surface. It's role in virulence and whether or not it is involved in host NK-cell avoidance is not understood. Further studies are needed on the cellular location of the SQPV MHC molecules and to clarify their possible role in avoiding NK-cell surveillance and immune evasion.
CD47 and anti-apoptosis.
The SQPV CD47 homolog shares 25–29% amino acid identity with a range of mammalian CD47 molecules and homology to the A38L protein found in Vaccinia virus (strain Copenhagen; VACV_Cop) and a range of other poxviruses. It shares strong sequence similarity to the immunoglobulin superfamily domain of CD47  (Figure S2; QMEANscore4 0.51) and is predicted to be membrane bound. Key cysteine residues associated with disulphide bond formation appear conserved whereas other residues critical for signal regulatory protein (SIRP) interaction are altered suggesting that SQPV CD47 may be structurally intact but fail to activate CD47 receptors.
In mammals, CD47 is widely expressed and its function is complex. It has been implicated in a range of T-cell regulatory functions including both inhibiting , and promoting  apoptosis, depending on its local molecular interactions. Deletion of the myxoma virus CD47 homolog (M128L) demonstrated that this gene is a virulence factor, necessary for lethal infections in susceptible rabbits, while it was fully dispensable for virus replication in vitro . Animals infected with the M128L-deleted virus showed greater activation of monocyte/macrophage cells in infected and/or lymphoid tissues.
The interferon system is activated in response to viral infection and plays an important role in the host defence system. Interferons released into the extracellular space bind interferon receptors on neighboring cells to induce an alert system. The receptors activate the JAK-STAT pathway and induce a range of interferon stimulated genes. Various poxviruses encode proteins that either bind (or are predicted to bind) interleukin-18 (IL-18), an inducer of type II IFN, or are mimics of both type I and type II IFN receptors . In the case of viral mimics they are thought to work by competitive inhibition, preventing the IFNs from binding to their natural receptors . Vaccinia virus (VACV) is also known to be able to inhibit the IFN-induced activation of STAT-1  whilst it is thought that MOCV may be able to inhibit IFN-induced NF-κB activation . SQPV does not seem to encode such genes but does encode a putative PKR inhibitor and, uniquely amongst the poxviruses, a 2′-5′ oligoadenylate synthetase (OAS)-like protein.
Protein Kinase (PKR).
Double-stranded RNA-activated protein kinase is a key component mediating the antiviral actions of IFN that restricts viral replication by phosphorylating eukaryotic initiation factor-2a-subunit, an initiator of protein synthesis, reducing levels of viral protein . Many viruses have evolved mechanisms to down regulate PKR function . The SQPV_025 is homologous to VACV_Cop E3L (41% identity over 69 amino acids toward N terminus), a host range gene, which is required for VACV pathogenesis . SQPV_025, VACV_Cop E3L and other poxvirus homologs share a small region of similarity in their carboxy terminus to an N-terminal domain of PKR (Figure 2). No other significant areas of homology exist between PKR and these poxvirus proteins. In vaccinia, this region has been shown to be responsible for inhibiting the activation of PKR both by binding to and sequestering dsRNA molecules , and by direct interaction with PKR itself .
Oligoadenylate synthetase (OAS).
OAS proteins are activated by dsRNA binding, leading to the OAS-catalysed synthesis of 2′-5′oligoadenylates (ppp(A2′p)n5′A, abbreviated to 2-5A) from ATP. RNase L, a latent endoribonuclease, becomes activated through binding 2-5As and degrades both viral and cellular RNA, including cellular rRNA . This antiviral response results in inhibition of protein synthesis and in some cases apoptosis. Protein alignments (Figure 3) show that the SQPV-OAS1 (SQPV_085) has a similar sequence to mammalian OAS1 proteins (128 identities out of 348 (37%) to porcine OAS [NP_999468]). The primary characteristics of the mammalian enzymes are: 2-5A synthesis, dsRNA binding, binding of other OAS enzymes, binding of 2-5As and ATP.
Coloured lines above the alignment indicates conserved functional regions and black lines indicates critical residues for function. Colours indicate different functional domains (Green, P-loop; Blue, PAFDAL; Yellow, LIRL; Peach, YALELLT and Pink, RPVILDPADP). The P-loop and PAFDAL domains hold the aspartic triad indicated by asterisks. The blue shading of the residues indicates the level of conservation, Dark blue is conserved in all species while light blue is conserved in just two. B. SQPV_0850 2′-5′ oligoadenylate synthetase – like molecule (blue) mapped to porcine 2′-5′ oligoadenylate synthetase OAS1 (PDB ID:1px5) (light yellow shadow); Green residues indicate the active DDD triad. Red residues indicate the equivalent residues RRE in the SQPV-OAS1 structure.
By in silico analysis, the SQPV-OAS1 gene is not conserved at amino acid residues known to be essential for normal OAS activity, lacking important aspartic triad residues  (Figure 3). A prediction of the tertiary structure based on the porcine OAS1 crystal structure (PDB ID:1px5) is shown in Figure 3b (QMEAN4 score 0.53). These observations suggested that SQPV-OAS1 protein should be capable of binding dsRNA, but is not expected to catalyse 2-5A synthesis. By doing so it may be that this viral protein is able to prevent or at least reduce the activation of RNase L.
It is clear from these results that SQPV is both in a novel taxonomic position in the chordopoxviruse subfamily which indicates a possible new genus of poxvirus, and that this virus has a previously undescribed diversity of virulence genes encoded in its genome. It would seem that this virus has adapted to it's host niche using genes commonly associated with pox virulence (e.g. CD47, PKR), but has also acquired novel genes such as multiple MHC molecules, and a way of potentially inhibiting the 2-5A system of the host.
The precise advantage and role of the specific virulence genes, and how they contribute to the difference in disease between grey squirrel populations in which it appears non-pathogenic and red squirrel populations in which it is emerging as lethal infection, is still to be determined.
Structural alignment of A. SQPV_004 (blue) aligned to class I MHC H-2Kk (light yellow) and β 2-microblobulin (red) with a peptide (green) in the peptide binding groove (based on PDB entry DOI:10.2210/pdb1zt7/pdb). Structural alignment of B. SQPV_027 (blue) aligned to class I MHC-like HFE (yellow) and β 2-microblobulin (red) (based on PDB entry DOI:10.2210/pdb1a6z/pdb) Structural alignment of C. SQPV_064 (blue) aligned to class I MHClike HFE (yellow) and β2-microblobulin (red) (based on PDB entry DOI:10.2210/pdb1a6z/pdb).
A. Predicted structure of SQPV_132 (VACV_Cop A38L) (Blue) with the extracellular domain of of CD47 (light yellow) and SIRPα (green) (based on PDB entry 10.2210/pdb2jjs/pdb). B. Alignment of SQPV_132 with CD47 antigen isoform 3 precursor [Homo sapiens] (NM_001025079.1. The cysteine residues that form a conserved disulphide bond between β sheets  are highlighted in yellow. Residues mediating specific polar interactions with SIRPa are in magenta.
Gene table function and vaccinia virus homology. The table shows the location and annotation of the predicted SQPV genes plus the predicted ortholog in other species. The “Nearest Poxvirus GeneID” column the ID of the gene with the top blast hit. The Product column gives the annotation of the predicted product based on the best blast hit.
Conceived and designed the experiments: ACD CJM ADR JC NH PMM. Performed the experiments: MH ARW KHK. Analyzed the data: NH ACD ADR CJM KHK. Contributed reagents/materials/analysis tools: CJM. Wrote the paper: CJM ACD AR NH JC.
- 1. Alcami A (2003) Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol 3: 36–50.
- 2. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201.
- 3. Brandt TA, Jacobs BL (2001) Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model. J Virol 75: 850–6.
- 4. Bruemmer CM, Rushton SP, Gurnell J, Lurz PW, Nettleton P, et al. (2010) Epidemiology of squirrelpox virus in grey squirrels in the UK. Epidemiol Infect 138: 941–50.
- 5. Buller RM, Palumbo GJ (1991) Poxvirus pathogenesis. Microbiological reviews 55: 80–122.
- 6. Cameron CM, Barrett JW, Mann M, Lucas A, McFadden G (2005) Myxoma virus M128L is expressed as a cell surface CD47-like virulence factor that contributes to the downregulation of macrophage activation in vivo. Virology 337: 55–67.
- 7. Chang HW, Jacobs BL (1993) Identification of a conserved motif that is necessary for binding of the vaccinia virus E3L gene products to double-stranded RNA. Virology 194: 537–47.
- 8. Delcher A, Harmon D, Kasif S, White O, Salzberg S (1999) Improved microbial gene identification with GLIMMER. Nucl Acids Res 27: 4636–4641.
- 9. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113.
- 10. García MA, Gil J, Ventoso I, Guerra S, Domingo E, et al. (2006) Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev 70: 1032–60.
- 11. García MA, Meurs EF, Esteban M (2007) The dsRNA protein kinase PKR: virus and cell control. Biochimie 89: 799–811.
- 12. Gardai SJ, Bratton DL, Ogden CA, Henson PM (2006) Recognition ligands on apoptotic cells: a perspective. J Leukoc Biol 79: 896–903.
- 13. Gil J, Rullas J, Alcam J, Esteban M (2001) MC159L protein from the poxvirus molluscum contagiosum virus inhibits NF-kappaB activation and apoptosis induced by PKR. The J Gen Virol 82: 3027–34.
- 14. Gubser C, Hu S, Kellam P, Smith GL (2004) Poxvirus genomes: a phylogenetic analysis. The J Gen Virol 85: 105–17.
- 15. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, et al. (2010) New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0. Systematic Biology 59: 307–21.
- 16. Haga IR, Bowie AG (2005) Evasion of innate immunity by vaccinia virus. Parasitology 130: S11–25.
- 17. Haller O, Kochs G, Weber F (2007) Interferon, Mx, and viral countermeasures. Cytokine Growth Factor Rev 18: 425–33.
- 18. Hatherley D, Graham SC, Turner J, Harlos K, Stuart DI, et al. (2008) Paired receptor specificity explained by structures of signal regulatory proteins alone and complexed with CD47. Mol Cell 31: 266–77.
- 19. Hovanessian AG, Justesen J (2007) The human 2′-5′oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2′-5′ instead of 3′-5′ phosphodiester bond formation. Biochimie 89: 779–88.
- 20. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, et al. (2009) InterPro: the integrative protein signature database. Nucl Acids Res 37: D211–215.
- 21. Kjaer KH, Poulsen JB, Reintamm T, Saby E, Martensen PM, et al. (2009) Evolution of the 2′-5′-oligoadenylate synthetase family in eukaryotes and bacteria. J Mol Evol 69: 612–24.
- 22. Lewis-Jones S (2004) Zoonotic poxvirus infections in humans. Curr Opin Infect Dis 17: 81–89.
- 23. Lukashin A, Borodovsky M (1998) GeneMark.hmm: new solutions for gene finding. Nucl Acids Res 26: 1107–1115.
- 24. McInnes CJ, Wood AR, Thomas K, Sainsbury AW, Gurnell J, et al. (2006) Genomic characterization of a novel poxvirus contributing to the decline of the red squirrel (Sciurus vulgaris) in the UK. J Gen Virol 87: 2115–2125.
- 25. McInnes CJ, Coulter L, Dagleish MP, Fiegna C, Gilray J, et al. (2009) First cases of squirrelpox in red squirrels (Sciurus vulgahs) in Scotland. Vet Rec 164: 528–531.
- 26. Moss B (2001) Poxviridae: the viruses and their replication., p. 2849–2883. In D. MKnipe and P. MHowley (ed.), Fields Virology, 4th ed. Lippincott Williams & Wilkins., Philadelphia.
- 27. Orange JS, Fassett MS, Koopman LA, Boyson JE, Strominger JL (2002) Viral evasion of natural killer cells. Nat Immunol 3: 1006–12.
- 28. Perdiguero B, Esteban M (2009) The interferon system and vaccinia virus evasion mechanisms. J Interferon Cytokine Res 29: 581–98.
- 29. Pettersen RD (2000) CD47 and death signaling in the immune system. Apoptosis 5: 299–306.
- 30. Rushton SP, Lurz PWW, Gurnell J, Nettleton P, Bruemmer C, et al. (2006) Disease threats posed by alien species: the role of a poxvirus in the decline of the native red squirrel in Britain. Epidemiol Infect 134: 521–533.
- 31. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, et al. (2000) Artemis: sequence visualization and annotation. Bioinformatics 16: 944–945.
- 32. Sainsbury AW, Deaville R, Lawson B, Cooley WA, Farelly SS, et al. (2008) Poxviral disease in red squirrels sciurus vulgaris in the UK: Spatial and temporal trends of an emerging threat. EcoHealth 5: 305–316.
- 33. Sainsbury AW, Nettleton P, Gilray J, Gurnell J (2000) Grey squirrels have high seroprevalence to a parapoxvirus associated with deaths in red squirrels. Animal Conservation 3: 229–233.
- 34. Scott AC, Keymer IF, Labram J (1981) Parapoxvirus infection of the red squirrel (Sciurus vulgaris). Vet Rec 109: 202.
- 35. Seet BT, Johnston JB, Brunetti CR, Barrett JW, Everett H, et al. (2003) Poxviruses and immune evasion. Annu Rev Immunol 21: 377–423.
- 36. Senkevich TG, Moss B (1998) Domain structure, intracellular trafficking, and beta2-microglobulin binding of a major histocompatibility complex class I homolog encoded by molluscum contagiosum virus. Virology 250: 397–407.
- 37. Sharp TV, Moonan F, Romashko A, Joshi B, Barber GN, et al. (1998) The vaccinia virus E3L gene product interacts with both the regulatory and the substrate binding regions of PKR: implications for PKR autoregulation. Virology 250: 302–15.
- 38. Smith VP, Bryant NA, Alcam A (2000) Ectromelia, vaccinia and cowpox viruses encode secreted interleukin-18-binding proteins. J Gen Virol 81: 1223–30.
- 39. Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56: 564–577.
- 40. Taylor JM, Barry M (2006) Near death experiences: poxvirus regulation of apoptotic death. Virology 344: 139–50.
- 41. Tompkins DM, Sainsbury AW, Nettleton P, Buxton D, Gurnell J (2002) Parapoxvirus causes a deleterious disease in red squirrels associated with UK population declines. Proc R Soc Lond [Biol] 269: 529–533.
- 42. Wauters LA, Lurz PWW, Gurnell J (2000) Interspecific effects of grey squirrels (Sciurus carolinensis) on the space use and population demography of red squirrels (Sciurus vulgaris) in conifer plantations. Ecol Res 15: 271–284.