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Short Communication |
1 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
2 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
Correspondence
David M. Haig
david.haig{at}moredun.ac.uk
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ABSTRACT |
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A supplementary figure is available with the online version of this paper.
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MAIN TEXT |
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140 kb double-stranded DNA prototype parapoxvirus, which causes orf (also known as contagious ecthyma or scabby mouth) after infection of damaged/scarified skin. The virus has a worldwide distribution and infects sheep, goats and man (Fleming & Mercer, 2006
; Haig & Mercer, 1998; Haig & McInnes, 2002). Infections are mainly acute, giving rise to local pustular lesions that turn to scabs. Immune evasion by orf virus is implicated because the virus can repeatedly infect previously exposed lambs in spite of an apparently normal host antivirus immune response (Anderson et al., 2001; Haig et al., 1992, 1996a, b, c). The orf virus genome has been sequenced (Delhon et al., 2004; Mercer et al., 2006). Several immuno-modulatory or host cell-derived genes have been identified (Fleming & Mercer, 2006). These include a viral orthologue of the ovine interleukin (IL)-10 gene (Fleming et al., 1997, 2000; Haig et al., 2002; Imlach et al., 2002; Lateef et al., 2003).
IL-10 is a pleiotropic cytokine that can exert either immuno-stimulatory or immuno-suppressive effects on many cell types (Moore et al., 2001). Orf virus IL-10 (vorfIL-10) is an early virus gene, the product of which is biologically active (Chan et al., 2006; Fleming et al., 1997; Haig et al., 2002; Imlach et al., 2002; Lateef et al., 2003). The orf virus polypeptide consists of 186 aa and a molecular mass of 21.7 kDa. It has 91 % amino acid identity to mature ovine IL-10. The C-terminal region encompassing two-thirds of the protein is identical to ovine IL-10. The N-terminal region shows little similarity with cellular IL-10 and in this respect resembles Epstein–Barr virus IL-10 (Vieira et al., 1991). We previously demonstrated that vorfIL-10 exhibited both anti-inflammatory and immuno-stimulatory activity in in vitro assays with ovine cells (Haig et al., 2002) and inhibited the maturation, antigen presentation and migration of murine dendritic cells (Lateef et al., 2003). Both vorfIL-10 and ovine cellular IL-10 suppressed ovine macrophage production of IL-8 and tumour necrosis factor (TNF)-
as well as blood mononuclear cell production of interferon (IFN)-
. In addition, either vorfIL-10 or ovine cellular IL-10 co-stimulated ovine and murine mast cell proliferation provided that IL-3 (for the ovine cells) or IL-4 (for the murine cells) was also present. The objective of this study was to determine if vorfIL-10 is a virulence factor by infecting groups of sheep with recombinant orf viruses lacking the vorfIL-10 gene (vorfIL-10ko virus) and vorfIL-10ko virus in which the vorfIL-10 gene was re-inserted (revertant virus, vorfrevIL-10), and comparing the clinical outcome (lesion size and time to resolution) to the wild-type parent virus (wt virus).
The vorfIL-10 gene knockout virus (strain NZ7) was made as described previously (Savory et al., 2000). The vorfIL-10 gene is located within the restriction endonuclease fragment EcoRI-D (Fleming et al., 1997; Robinson et al., 1987). A 494 bp fragment which spans 211 bp downstream of the vorfIL-10 coding region, and which includes the terminal 283 bp of the vorfIL-10 gene (right arm), was PCR amplified and cloned into the vector pSP70 at the EcoRI and BglII restriction sites (pSP70/right arm). A 517 bp fragment that spans 391 bp upstream of the vorfIL-10 coding region, and which includes the first 126 bp of the vorfIL-10 gene (left arm), was PCR amplified and cloned into the HindIII and EcoRI restriction sites of pSP70/right arm. Finally, the Escherichia coli 
-galactosidase gene (lacZ) under the control of an orf virus late promoter PFI (Fleming et al., 1993) was cloned as a reporter gene into an EcoRI site located between the left and right arms. The construct was designed to delete 149 nt from the coding sequence of the vorfIL-10 gene and replace this sequence with -galactosidase. The final plasmid construct was called pIL-10NZ7-
. Recombinant orf virus (vorfIL-10ko) was generated using a procedure adapted from standard protocols used in the generation of vaccinia virus recombinants (Mackett et al., 1984) and methods described previously (Savory et al., 2000). Putative recombinant plaques were identified 4–5 days after infection by their blue phenotype in the presence of 5-bromo-4-chloro-3-indoyl -D-galactopyranoside (X-Gal). Characterization of the vorfIL-10ko virus was carried out prior to the construction of the vorfrevIL-10 recombinant and is described below.
To construct the vorfrevIL-10 recombinant, a 974 bp fragment spanning 391 bp upstream of the vorfIL-10 coding sequence and 33 bp downstream of the vorfIL-10 gene (left arm) was PCR amplified and cloned into the HindIII and EcoRI restriction sites of pSP70. A 466 bp fragment spanning a region downstream of the vorfIL-10 gene and beginning 37 bp downstream from the vorfIL-10 coding sequence was PCR amplified and cloned into the vector pSP70/left arm at the EcoRI and BglII restriction sites. A synthetic poxvirus early/late promoter was inserted into the EcoRI site immediately downstream of the IL-10 gene and between the left and right arms. The synthetic promoter also provided a transcription termination sequence for the vorfIL-10 gene. The reporter gene -glucuronidase gene (GUS) was then inserted into an XbaI site immediately downstream of the synthetic promoter. This placed the GUS gene downstream of vorfIL-10 in an intergenic region in the vorfrevIL-10 recombinant. In addition an early transcription termination sequence, TTTTTCT, was engineered immediately downstream of the GUS gene. The final plasmid construct was called pIL-10NZ7restore. The recombinant virus (vorfrevIL-10) was generated from the vorfIL-10ko recombinant by methods described above. Putative vorfrevIL-10 recombinant virus plaques were identified by their blue phenotype in the presence of 5-bromo-4-chloro-3-indolyl -D-glucuronide cyclohexylammonium salt and lack of blue colour in the presence of X-Gal.
Characterization of vorfIL-10ko and vorfrevIL-10 was carried out by restriction endonuclease digests and of viral genomic DNA and PCR amplification of specific regions. The unique EcoRI and BamHI fragments produced for vorfIL-10ko and vorfrevIL-10 viruses matched those predicted for the recombinants (Fig. 1). PCR amplification showed that the 149 bp region was deleted from the recombinant but present in the vorfIL-10 virus. Table 1 shows that the wt virus and the vorfrevIL-10 virus expressed vorfIL-10 in culture, whereas the vorfIL-10ko virus did not. Interestingly, the levels of vorfIL-10 produced from orf virus-infected cells were comparable to physiologically relevant levels of cellular IL-10 that have been estimated to be approximately 0.5 to 5 ng ml–1 (Chang et al., 2004).
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For the in vivo experiments, virus–dose responses in primary infection were performed using orf-virus-naïve (seronegative) merino-cross lambs (all animal experiments described in this study were approved by the University of Otago, Animal Ethics Committee). Animals were divided into three groups of four and inoculated on the wool-free region of the inner hind legs. Group 1 was infected with wt orf virus strain NZ7 (wt virus), group 2 with vorfIL-10ko virus and group 3 with vorfrevIL-10 virus. Four lines of scarification of approximately 5 cm in length were made on the epidermis of each of the hind legs and infected by topical application of 100 µl per line of PBS containing 1x108, 1x106 and 1x104 p.f.u. virus ml–1 or with PBS only (no virus control). Virus-induced lesions were photographed daily over a 3-week period and the clinical scores determined. For reinfection experiments, four groups of six animals were infected with the wt virus, vorfIL-10ko virus, vorfrevIL-10 virus and PBS only as described above. The animals had received 106 wt virus per scar line 3 months previously as a primary infection.
Sheep were examined for erythema, vesicle and pustule formation and the presence of a firmly attached scab associated with the scarified/infected areas of the skin. Clinical scores were calculated as described previously (Nettleton et al., 1996). For statistical analysis, a one-way ANOVA test was applied to data normalized by log10 transformation. The confidence limit was set at 95 %. Values of P<0.05 were considered significant.
Fig. 2(a–d) shows the time course of lesion development as measured by clinical scores in animals receiving vorfIL-10ko virus, vorfrevIL-10 virus and wt virus. Fig. 2(d) shows in addition the clinical score of control animals receiving PBS on scar lines. In primary infection (Fig. 2a–c) with three different doses of the orf viruses, the vorfIL-10ko virus-induced orf lesions of lower magnitude in terms of the disease severity (clinical score) compared with wt or vorfrevIL-10 virus. There was evidence of a virus dose-dependent clinical response, with more virus (wt, vorfrevIL-10 or vorfIL-10ko) in the infection inoculum associated with larger, more severe lesions, as has been previously shown for wt virus (Haig et al., 1996d; Haig & Mercer, 1998). vorfIL-10ko virus attenuation was demonstrated, showing significantly reduced clinical scores (P<0.02) when compared with wt virus at either 106 or 108 virus per inoculum on all days after infection. There was also a significant difference between vorfIL-10ko and vorfrevIL-10 virus groups with the 106 and 108 virus doses on all days after infection (P<0.04, Fig. 2b). The vorfrevIL-10 revertant virus did not induce lesions as severe as those induced by wt virus at the same dose, but this difference was not significant (P>0.05). In the 104 virus inoculum group (Fig. 2a), vorfIL-10ko virus infection gave a significantly lower lesion score only on day 10 after infection (P<0.03) when compared with both the wt virus and the vorfrevIL-10 virus-infected groups. Fig. 2 also shows a comparison of lesion growth for the viruses at 7 days post-infection. A 100–1000-fold greater dose of the vorfIL-10ko virus was required to induce lesions with a clinical score equivalent to the wt and vorfrevIL-10 viruses, and the vorfIL-10ko virus showed little growth at the lowest dose. This suggests that the vorfIL-10 gene is important in the establishment of infection by orf virus and that innate responses (e.g. IFNs and TNF) could be important in inhibiting orf virus replication during the early stages of infection.
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), vorfIL-10ko virus infection was associated with smaller lesions over the time course of the experiment compared with the vorfIL-10 intact viruses. Statistically significant differences were seen for all time points between vorfIL-10ko and wt virus (P<0.04), and between vorfIL-10ko and vorfrevIL-10 viruses on all days (P<0.03) except day 2. There was no significant difference between the vorfrevIL-10 virus and wt virus clinical scores at any time point (P>0.05).
The results demonstrate that orf virus IL-10 is a virulence factor, as virus lacking the vorfIL-10 gene was attenuated in primary infections and reinfections of sheep compared with virus with intact vorfIL-10 as measured by differences in clinical scores. Clinical scores of lesion development provide an accurate readout of the direct consequence of virus infection and replication, and reflect viral load (Haig et al., 1996d). Reinsertion of vorfIL-10 in orf virus (vorfrevIL-10 virus in reinfections) was successful in largely restoring the wt phenotype, confirming that the attenuation of the virus was due to a lack of vorfIL-10. Although not significant, the clinical score for the vorfrevIL-10 virus was generally less than that for wt virus. This could have been due to the presence of the reporter gene in the revertant virus or an additional change in the wt virus compared with the vorfIL-10ko virus that was retained in the revertant.
Previously, we had demonstrated that vorfIL-10 acted as an early gene during the virus life cycle with properties similar to that of cellular IL-10 (Haig et al., 2002). This was in spite of vorfIL-10 differing substantially in amino acid sequence within the N-terminal third of the molecule. vorfIL-10 inhibited the transcription and expression of ovine IFN- and the chemokine IL-8, and to a lesser extent TNF- and IL-1. We had also previously shown, with wt orf virus, that cell recruitment to the region of infection increased and declined in parallel with the increase and decrease of lesion size that also itself correlated with virus load in the epidermis (Haig et al., 1996d). In the present study, this was generally the case – for example, declining cell numbers in the vorfIL-10ko-infected animals compared with wt virus-infected animals between days 15 and 20 of a primary infection (haematoxylin and eosin analysis not shown). Future work will determine whether the vorfIL-10 is acting on dendritic cell function and/or suppression of inflammatory cytokines (e.g. IFN, TNF).
In conclusion, vorfIL-10 is a virulence factor. The vorfIL-10 is one of several known virulence or immuno-modulatory factors expressed by the virus. There are more putative immuno-modulatory genes yet to be characterized in the flanking region of the orf virus genome (Delhon et al., 2004; Fleming & Mercer, 2006; Mercer et al., 2006; Wood & McInnes, 2003). It is probable that all of the virulence/immuno-modulatory factors act in concert to suppress the immune response to orf virus, facilitate infection of epidermal keratinocytes and allow the virus to replicate and shed prior to clearance by the host immune response. Removal of any one of these genes would be expected to have only a partial effect on virulence, which is what was observed in this study with vorfIL-10.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 January 2007;
accepted 23 February 2007.
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, wt virus; 
, vorfIL-10ko virus; 
, vorfrevIL-10 virus; 
, PBS control. Asterisks indicate where the vorfIL-10ko virus clinical score is significantly (P<0.04) different from that of the wt virus and the vorfrevIL-10 virus. (e–g) Orf virus lesions induced after primary infection by the wt virus, vorfIL-10ko virus and vorfrevIL-10 virus, respectively, on day 7 after primary infection (lesions shown by arrowheads). The top lesion in each case was after 108 p.f.u. virus ml–1, the middle ones after 106 p.f.u. virus ml–1 and the bottom ones after 104 p.f.u. virus ml–1.