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1 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
2 Institute of Zoology, London, UK
3 Queen Mary, University of London, UK
4 National Wildlife Health Center, Madison, WI, USA
Correspondence
Colin J. McInnes
Colin.mcinnes{at}moredun.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Published online ahead of print on 24 April 2006 as DOI 10.1099/vir.0.81966-0
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ377804 and DQ377805.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The decline of the red squirrel has been attributed to the introduction of the North American grey squirrel (Sciurus carolinensis) because of its ability to successfully out-compete the red species for habitat and food. However, more recently a lethal epidemic disease that is characterized by severe ulceration and formation of haemorrhagic scabs around the eyes, nose and mouth was suggested as an additional reason for the local extinction of red squirrel populations (Sainsbury & Gurnell, 1995; Sainsbury et al., 2000). The putative cause of the disease was identified as a poxvirus and based on its morphological similarity with viruses belonging to the family Parapoxvirinae, it was classified and named Squirrel parapoxvirus (SPPV) (Scott et al., 1981; Buller et al., 2005). Recently, we reported on the phylogenetic analysis of the genes encoding the viral major membrane protein and the 30 kDa subunit of the viral RNA polymerase. This analysis did not support the classification of the virus as a parapoxvirus, but instead suggested that it most likely represents a previously unidentified genus within the poxvirus family. As a consequence, we suggested that the name of the virus should be changed from SPPV to squirrelpox virus (SQPV; Thomas et al., 2003).
Experimental infections of squirrels with SQPV confirmed that it is the agent responsible for the disease seen in wild red squirrels and that conversely, the grey squirrel, although infected by the virus, is apparently clinically resistant to the disease (Tompkins et al., 2002; Thomas et al., 2003). A serological survey of squirrels sampled from across the UK demonstrated that 61 % of grey squirrels had antibodies to SQPV in contrast to only 2.9 % of red squirrels, the overwhelming majority of which were found dead or dying of the disease (Sainsbury et al., 2000). The high seroprevalence of antibodies to the virus, but absence of disease, in the grey squirrel suggests that they may act as a reservoir host for the virus, transmitting it to susceptible red squirrels with lethal consequences, a theory that is supported by transmission studies and mathematical modelling of the dynamics of local squirrel populations (Tompkins et al., 2003; Rushton et al., 2005). Outbreaks of pox-like disease in red squirrels were not reported until after the introduction of the grey squirrel from America and it is thought that the virus was introduced to Britain via this route, although the virus has never been described in the USA. There is, however, the possibility that the virus is endemic to the UK and that other rodent species inhabiting the same woodland environment could be harbouring the virus.
The poxvirus family consists of two subfamilies, the Chordopoxvirinae and the Entomopoxvirinae. The subfamily Chordopoxvirinae can be further divided into eight genera based mainly on the animal species they infect, but also on virion morphology, antigenic cross-reactivity, genome size and gene content. Their genomes are linear double-stranded DNA molecules ranging in size from approximately 135 to over 300 kb. Within them 89 genes from the central ‘core’ of the genomes are conserved across all sequenced species, whereas the ‘termini’ vary considerably in size and gene content between the genera (Upton et al., 2003; Gubser et al., 2004; Delhon et al., 2004). Successful vaccination against a particular poxvirus is usually only possible using the same, but generally attenuated, virus or one from the same genus. Cross-genera vaccination is not normally successful. Transmission of the viruses also differs between genera, being spread either by aerosol, contaminated fomites, direct contact or arthropods. It was important for us to determine the classification of SQPV, because many of the assumptions previously made about the transmission and survivability of the virus and its potential for vaccine development, were based on its original classification as a Parapoxvirus. Here, we present a genetic map of SQPV and approximately 23 and 37 kb of contiguous sequence from the left-hand (LH) and right-hand (RH) termini, respectively. The gene content and genomic organization of SQPV is compared with Orf virus (ORFV), the prototypic parapoxvirus and other poxviruses. Phylogenetic analysis of 15 genes conserved across all the recognized genera of the subfamily Chordopoxvirinae is used to infer classification of the virus, whilst further serological studies failed to find evidence of the virus in other British woodland rodents, but provided preliminary evidence that the virus is present in grey squirrels in the USA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Isolation and cloning of viral genomic DNA.
Virus DNA was isolated from scabs collected post-mortem. These were homogenized in PBS and centrifuged at 2000 g for 30 min at 4 °C. The viral cores were purified from the resulting supernatant by centrifugation through a sucrose cushion [36 % (w/w) in PBS] at 71 000 g for 30 min at 4 °C. The SQPV genomic DNA was subsequently extracted and purified from these cores (Gilray et al., 1998). The purified DNA was digested with the restriction endonucleases NotI, KpnI and BamHI (Roche Diagnostics) and the resulting fragments cloned into the pBluescript SK– plasmid vector (Stratagene). In addition, viral genomic DNA was partially digested with Sau3AI (Roche Diagnostics) and the resulting fragments, 30–40 kb in size, were cloned into the SuperCos I cosmid vector (Stratagene).
Restriction endonuclease mapping of SQPV genomic DNA cloned into SuperCos I.
Restriction endonuclease maps of cosmid clones were determined using a standard procedure (Rackwitz et al., 1985). Briefly, cosmid clones were linearized by digestion with 
-terminase (Amersham Pharmacia Biotech). The linearized cosmid clones were partially digested with the desired restriction enzyme and labelled at one end by hybridization in solution with one or other of the 32P-labelled oligonucleotides complementary to the 12 bp cos site. The labelled reactions were electrophoresed on a 0.5 % (w/v) agarose gel, the gel was dried onto DE-81 chromotography paper and exposed to X-ray film. The restriction maps were determined from the fragment sizes of the partially digested, end-labelled cosmid DNA.
Sequence analysis.
The EZ : : TN<KAN-2> Insertion kit (Epicentre) was used to randomly introduce a kanamycin resistance marker and primer binding sites into plasmid clones containing SQPV fragments. Equimolar amounts of target DNA and <KAN-2> transposon were mixed in vitro in transposition insertion reactions. An aliquot of each reaction mixture was used to transform One Shot TOP10 Escherichia coli (Invitrogen) and transformants were selected on agar plates containing 50 µg kanamycin ml–1. Double-stranded DNA templates from a selection of transformants for each of the SQPV clones were prepared and sequenced using KAN-2 FP-1 and KAN-2 RP-1 primers that correspond to the primer binding sites found on the transposon. Sequencing reactions were run on a GE Healthcare MegaBACE 500 capillary DNA sequencer using ‘DYEnamic’ ET Terminator chemistry (Amersham Pharmacia). DNA sequences were compared to those deposited in the GenBank/EMBL database using the FastA 3 search algorithm (Pearson & Lipman, 1988). In addition, DNA sequences were translated into all six open reading frames (ORFs) using the DNASTAR package (DNASTAR) and the predicted amino acid sequences compared with those deposited in protein databases. Further sequence analyses were performed using the VOCS website (Ehlers et al., 2002) and the ARTEMIS sequence analysis package (Rutherford et al., 2000).
DNA hybridization.
Double-stranded DNA probes were labelled with digoxigenin (DIG) using the nick-translation labelling kit (Roche Diagnostics). Digested viral genomic DNA was transferred from agarose gels and immobilized onto Hybond-N membrane (Amersham Pharmacia Biotech). Alternatively, 0.5 µg heat-denatured cloned DNAs were applied to Hybond-N membranes using a Hybri-dot vacuum manifold. Membranes were hybridized with the DIG-labelled probes at 65 °C for approximately 16 h, before washing. Hybridization was detected with anti-DIG antibody conjugated to alkaline phosphatase using the manufacturer's recommended procedures.
Direct ELISA.
An adaptation of the ELISA described by Sainsbury et al. (2000) was used to detect anti-SQPV IgG in the sera from both red and grey squirrels and from wood mice and bank voles. Briefly, duplicate wells in 96-well microplates were coated overnight at 4 °C with 50 µl SQPV, and negative control, antigens. After removal of unbound antigen, test and control sera were applied, in duplicate, to wells coated with positive- and negative-antigen and incubated at 37 °C for 1 h. After washing, 100 µl protein G-horseradish peroxidase conjugate diluted 1/750 in ELISA dilution buffer [1x PBS with 1 % (w/v) BSA (Sigma)], was applied to each well and incubated at 37 °C for 1 h. After further washing, 100 µl freshly prepared orthophenylene diamine (Sigma) substrate was added to each well. The colorimetric reaction was allowed to proceed for approximately 8 min before the OD492 of each well was determined and the corrected optical densities for each test and control serum calculated by subtracting the mean optical density of the negative-antigen wells. An OD492 value of 0.2 was used as the cut-off value to discriminate between positive- and negative-antibody detection.
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RESULTS |
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) with the corresponding sizes of the restriction fragments summarized in Table 1. The terminal restriction fragments were not cloned, but are presumed to contain the terminal hairpin loop structures found at the ends of all poxvirus genomes. However, using a Southern blot of SQPV genomic DNA digested with KpnI and hybridized with BamHI fragment B' (not shown), the sizes of the terminal KpnI fragments J and D were estimated to be 5.3 and 9.0 kb, respectively. As a result the positions of the two ends of the genome could be estimated. The sum of the sizes of restriction fragments generated with a single enzyme indicated that the SQPV genome is approximately 158 kb in length, whilst targeted sequencing of BamHI restriction fragments K and V suggested that the inverted terminal repeat (present in all poxviruses) was approximately 5 kb in length.
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. Although many of the genes could be recognized as orthologues of known poxvirus genes, others, particularly at the extreme ends of the genome, did not appear to have counterparts in other poxviruses and indeed did not appear to be similar to anything in the sequence databases. These sequences were subjected to further analysis to determine whether or not they contained recognizable elements that would suggest their properties and/or function. A summary of these analyses is presented in Table 3.
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. Using Fowlpox virus (FWPV) as an outgroup four major groupings (including FWPV) are clearly delineated with SQPV partitioning with the other viruses containing a genome base composition of >60 % G+C residues. However, there is 100 % bootstrap support for the branch that separates the SQPV from both MOCV and the parapoxviruses, suggesting that SQPV diverged before the line that gave rise to MOCV and the parapoxviruses.
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. The positive-control samples (seropositive grey squirrel sera) were consistently positive (corrected optical density values ranged from 1.223 to 1.482) and the negative controls (seronegative grey squirrel sera and BALB/c mouse) all had corrected optical density values less than 0.1. Likewise, all the corrected optical density values for the wood mice and bank vole sera were less than 0.1, suggesting that these animals, at least, had not been infected by the virus. Serum samples were also collected from seven grey squirrels trapped in Dane County, Wisconsin, USA, and tested by ELISA. All seven serum samples tested positive, with readings ranging from 0.59 to 2.78 (median=2.07). This is the first time antibodies to SQPV have been detected in serum samples originating from the USA. |
DISCUSSION |
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; Becher et al., 2002; Thomas et al., 2003; Tikkanen et al., 2004). The restriction mapping reported here suggests a genome size of approximately 158 kb, nearly 20 kb longer than any wild-type ORFV genomes previously reported and 25 kb longer than the Bovine papular stomatitis virus (BPSV) genome, these being the only two parapoxviruses for which there is full genomic sequence published (Menna et al., 1979; Robinson et al., 1982, 1987; Mercer et al., 1987, 2006; McInnes et al., 2001; Delhon et al., 2004). Some of the extra length is due to there being approximately 8 kb of extra sequence between the end of the orthologue of the VACV-Cop E4L gene and the ITR at the LH end of the genome in SQPV when compared to ORFV. Part of the reason for this is that the translocation of the orthologues of the VACV F9L and F10L genes from the LH end of the genome to the RH end, which appears to be characteristic of the genus Parapoxvirinae (Mercer et al., 1995; Ueda et al., 2003; Rziha et al., 2003; Delhon et al., 2004), has not occurred in the SQPV genome. Instead these genes are found in a location similar to that in all the other chordopoxvirus genera. Detailed sequence analysis of the two ends of the genome also revealed little similarity between the SQPV genome and those of the genus Parapoxvirinae. In particular none of the genes such as the vVEGF, the vIL-10 or the ‘ankyrin-like’ repeat genes found in both ORFV and BPSV are found in the corresponding regions of the SQPV genome. Indeed SQPV is the only virus, other than MOCV, that, in the terminal regions of its genome, does not encode proteins predicted to contain ankyrin-like repeats.
The majority of poxvirus genes found between the F9L gene (VACV-Cop nomenclature) at the LH end of poxvirus genomes and the A34R gene at the RH end are essential for replication of the viruses in vivo and in vitro, whereas those outwith this region are generally considered non-essential, conferring instead an advantage to the viruses in combating an immune response or dictating the range of hosts that the viruses can successfully infect (Upton et al., 2003; Gubser et al., 2004). Many, but not all, of the genes found near the termini are genera-specific. In the SQPV genome, the majority of genes found near the left and right termini have no recognizable counterparts in other poxviruses. The remainder may be orthologues of known poxvirus genes, but in many of these instances the genes are found in different locations from the other poxviruses and the similarities between the proteins, predicted to be encoded by these genes, and their possible orthologues are extremely low.
At the LH end of the SQPV genome we have predicted that there are nine genes between the ITR and the orthologue of the F9L gene. Six of these, namely K1L, K2L, I3L, I4L, I6L and A1L (nomenclature based on the BamHI restriction fragment in which the start codon is located) have no counterparts in other poxviruses. Indeed they do not appear to be similar to any known sequences. The three remaining genes, I1L, I2L and I5L, are similar to three genes found only in MOCV. I1L appears to be similar in sequence to MOCV 003L and is found in the same relative position (Senkevich et al., 1997). MOCV 003L has a paralogue at the RH end of the MOCV genome, namely 157R, but the function of both remains obscure. A similar paralogous gene is found at the RH end of the SQPV genome (named W2R); however, the protein it encodes has slightly more similarity to the MC003L protein than the MC157R protein, suggesting that SQPV I1L might represent an ancestral gene, which was duplicated and translocated to the RH end of the genome and that subsequently in MOCV diverged to obtain properties or a function relevant to that particular virus. The SQPV I2L gene may be an orthologue of the MOCV 080R gene that encodes a protein with similarity to class I major histocompatibility complex (MHC) proteins (Senkevich & Moss, 1998). The predicted proteins encoded by each gene share a high degree of identity, but positionally the genes are found in quite different locations. Pairwise alignment of the viral proteins suggest they share approximately 21 % identity, but a similar analysis between the SQPV protein and a human MHC class I E protein suggests they share 39 % identity. This raises the question as to whether the two viral genes are in fact orthologues of each other, or whether the genes have been acquired by the two viruses independently. The human MHC class I E is a non-polymorphic molecule that is involved in signalling to natural killer (NK) cells the extent of MHC class I expression on a cell surface (Braud et al., 1998). Some poxviruses are known to downregulate the expression of MHC class I at the cell surface thus making these cells susceptible to NK-mediated cytolysis. It may well be that the SQPV protein may function to subvert this NK cell-mediated killing of virus infected cells. MHC orthologue genes are also found in the genomes of Yaba-like disease virus (YLDV) and Swinepox virus (SWPV) but neither of these are predicted to encode a transmembrane anchor sequence, thus distinguishing them from the MOCV and SQPV genes (Lee et al., 2001; Afonso et al., 2002). The last SQPV gene in this region, I5L, is similar to, and found in a similar relative position as, the MOCV 008L gene, the protein encoded by which is of unknown function (Senkevich et al., 1996, 1997).
In addition to the six genes found to the left of the F9L gene that had no counterparts in other poxviruses, a further three genes at the LH side of the SQPV genome, A7L, A8L and A9L, located between the orthologues of the VACV F13L and F15L genes also appear to be unique to SQPV. Using InterProScan (Zdobnov & Apweiler, 2001) to suggest possible functions for the proteins encoded by the ‘unique’ genes led to no predictions.
At the RH end of poxvirus genomes there is a region that in Cowpox virus (CPXV), amongst others, encodes the major protein component of the acidophilic-type inclusion (ATI) body associated with infected cells (Funahashi et al., 1988; Meyer & Rziha, 1993). The corresponding region in the other poxviruses varies considerably in size such that the gap between the orthologues of the VACV-Cop A24R and A27L genes ranges in size from approximately 450 bases in Yaba monkey tumour virus, SWPV and Lumpy skin disease virus to approximately 5850 bases in CPXV. The corresponding region in the SQPV genome is over 8200 bases in length. There are predicted to be three highly related genes within this region, the functional significance of which is unknown.
We have predicted that between the orthologue of VACV-Cop A34R and the ITR at the RH end of the SQPV genome there are 19 genes covering approximately 18 kb of sequence. It is likely that four of these are orthologues of the VACV-Cop genes A35R, A37R, A41L and A51R. There are a further nine SQPV genes, namely C3R, C7L, C10R, C14R, C15R, C16R, X1R, X2R and W2R that have some similarities to other poxvirus genes, but whether or not they are true orthologues of these genes remains to be determined. This is because in general poxvirus genomes have been shown to be collinear, whereas seven of nine genes above are found in a different genomic location from the corresponding gene in other poxviruses. For example, the SQPV C15R gene is predicted to encode a protein with 31 % identity to the 014 protein encoded at the opposite end of the Myxoma virus genome (Cameron et al., 1999), whilst the SQPV C10R has some sequence and positional similarity to the VACV A38L protein, but it is transcribed from the opposite strand of DNA to the VACV protein (Johnson et al., 1993). It may be that rather than representing true orthologues SQPV has evolved independently to encode a variety of proteins with functions similar to those in other poxviruses. As with the LH end of the genome there are at least six genes at the RH end, C4R, C6R, C8R, C11R, C12R and W1R that encode proteins with no similarity to anything in the databases. Analysis with InterProScan revealed little about their potential function.
No evidence has been found that would support the classification of SQPV as a parapoxvirus. The lengths and gene content of the non-conserved regions at the LH and RH sides are quite different from the parapoxviruses, with none of the genes considered to be characteristic of the parapoxviruses being found in the SQPV genome. In addition, not only does the phylogenetic analysis with the 15 proteins conserved across all of the subfamily Chordopoxvirinae suggest a classification separate from the parapoxviruses, the nearest poxvirus match for the remaining proteins, as judged by individual pairwise alignments, is rarely with the parapoxvirus orthologue. Indeed there is no discernable pattern as to which species or genera usually provides the closest poxvirus match. These results provide fresh impetus to the study of SQPV and the ways in which it differs from the other poxviruses. Previous assumptions about the virus, particularly the mode of transmission, based on it being a parapoxvirus should be readdressed.
The fact that antibody to SQPV was not detected in serum from wood mice and bank voles in two areas of the UK where grey squirrels are seropositive does not preclude the virus from being endemic within an, as yet, unidentified British wildlife species. However, with the serum samples collected from grey squirrels in Wisconsin, USA, being found to contain antibody against SQPV, the serological evidence, for the first time, supports the theory that the virus was introduced to the UK with the grey squirrel.
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ACKNOWLEDGEMENTS |
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Received 21 February 2006;
accepted 3 April 2006.
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