Therapeutic immunomodulation using a virus--the potential of inactivated orf virus.

Viruses can manipulate the immune response against them by various strategies to influence immune cells, i.e. by over-activation leading to functional inactivation, bypassing antigen presentation or even suppression of effector functions. Little is known, however, about how these features of immune regulation and modulation could be used for therapeutic purposes. Reasons for this include the complexity of immune regulatory mechanisms under certain disease conditions and the risks that infections with viruses pose to human beings. The orf virus (ORFV), a member of the Parapoxvirus genus of the poxvirus family, is known as a common pathogen in sheep and goats worldwide. The inactivated ORFV, however, has been used as a preventative as well as therapeutic immunomodulator in veterinary medicine in different species. Here, we review the key results obtained in pre-clinical studies or clinical studies in veterinary medicine to characterise the therapeutic potential of inactivated ORFV. Inactivated ORFV has strong effects on cytokine secretion in mice and human immune cells, leading to an auto-regulated loop of initial up-regulation of inflammatory and Th1-related cytokines, followed by Th2-related cytokines that attenuate immunopathology. The therapeutic potential of inactivated ORFV has been recognised in several difficult-to-treat disease areas, such as chronic viral diseases, liver fibrosis or various forms of cancer. Further research will be required in order to evaluate the full beneficial potential of inactivated ORFV for therapeutic immunomodulation.

Delivery of Echinococcus granulosus antigen EG95 to mice and sheep using recombinant vaccinia virus.

The tapeworm Echinococcus granulosus is the causative agent of hydatid disease and affects sheep, cattle, dogs and humans worldwide. It has a two-stage life cycle existing as worms in the gut of infected dogs (definitive host) and as cysts in herbivores and humans (intermediate host). The disease is debilitating and can be life threatening where the cysts interfere with organ function. Interruption of the hydatid life cycle in the intermediate host by vaccination may be a way to control the disease, and a protective oncosphere antigen EG95 has been shown to protect animals against challenge with E. granulosus eggs. We explored the use of recombinant vaccinia virus as a delivery vehicle for EG95. Mice and sheep were immunized with the recombinant vector, and the result monitored at the circulating antibody level. In addition, sera from immunized mice were assayed for the ability to kill E. granulosus oncospheres in vitro. Mice immunized once intranasally developed effective oncosphere-killing antibody by day 42 post-infection. Antibody responses and oncosphere killing were correlated and were significantly enhanced by boosting mice with either EG95 protein or recombinant vector. Sheep antibody responses to the recombinant vector or to EG95 protein mirrored those in mice.

Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins.

The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein. However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the BPSV protein.

Recurrent localised cutaneous parapoxvirus infection in three cats.

CASE HISTORY: Three cats were presented with single proliferative lesions affecting one foot, which failed to heal after medical treatment, and recurred despite surgical resection. PATHOLOGICAL FINDINGS: Histologically, the lesions were proliferative and papillary. There was marked acanthosis, rete peg formation, and compact orthokeratosis, with large numbers of bacteria in the orthokeratotic scale. Some biopsies had multifocal keratinocyte swelling of the stratum granulosum, and amphophilic intracytoplasmic inclusions were present in some of the swollen cells. The dermis consisted of a light fibrous stroma with marked capillary proliferation. Parapoxviruses were detected in the lesions of all cats by electron microscopic examination. PCR analysis detected orf virus (contagious ecthyma virus) in two cats, and orf virus was cultured from one cat. DIAGNOSIS: Parapoxvirus infection in cats. CLINICAL RELEVANCE: Parapoxvirus infection should be considered as a differential diagnosis when dealing with proliferative, non-healing lesions on the feet of cats, especially cats in rural areas. The recovery of orf virus from a cat with typical poxvirus lesions extends the range of species affected by this virus.

Construction of a recombinant orf virus that expresses an Echinococcus granulosus vaccine antigen from a novel genomic insertion site.

The potential of recombinant poxviruses as expression vectors has been extensively studied using Vaccinia virus but there has been only limited transfer of this technology to the Parapoxvirus genus. We detail here the construction of a recombinant Orf virus that expresses an antigenic peptide (EG95) of the causative agent of cystic hydatid disease, Echinococcus granulosus. Expression of this foreign antigen was regulated by a synthetic early/late poxvirus transcriptional promoter and levels of expression comparable to that achieved by a similar vaccinia virus recombinant were observed. The expression cassette was inserted into a unique orf virus gene (G1L) thereby confirming the non-essential nature of that gene and identifying a novel genomic insertion site. This recombinant will be a valuable tool with which to assess the potential of recombinant orf viruses to deliver vaccine antigens to sheep.

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure.

The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41.1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

Viral vascular endothelial growth factor plays a critical role in orf virus infection.

Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced.

Parapoxviruses are strongly inhibited in vitro by cidofovir.

Three parapoxviruses which cause orf or related diseases in humans and animals and the orthopoxvirus, vaccinia virus, were tested for their in vitro sensitivity to cidofovir. The 50% inhibitory concentration for the three parapoxviruses was between 0.21 and 0.27 microg/ml and for vaccinia was 1.32 microg/ml. The selectivity index varied from 198 to 264 for the parapoxviruses and was 42 for vaccinia virus. Virus yield assays confirmed the ability of cidofovir to reduce ortho- and parapoxvirus replication. The efficacy of cidofovir against parapoxviruses justifies its evaluation as a candidate drug for the treatment of parapoxvirus infections in humans and animals.

Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1.

Orf virus, a member of the poxvirus family, produces a pustular dermatitis in sheep, goats, and humans. The lesions induced after infection with orf virus show extensive proliferation of vascular endothelial cells, dilation of blood vessels and dermal swelling. An explanation for the nature of these lesions may lie in the discovery that orf virus encodes an apparent homolog of the mammalian vascular endothelial growth factor (VEGF) family of molecules. These molecules mediate endothelial cell proliferation, vascular permeability, angiogenesis, and lymphangiogenesis via the endothelial cell receptors VEGFR-1 (Flt1), VEGFR-2 (KDR/Flk1), and VEGFR-3 (Flt4). The VEGF-like protein of orf virus strain NZ2 (ORFV2-VEGF) is most closely related in primary structure to VEGF. In this study we examined the biological activities and receptor specificity of the ORFV2-VEGF protein. ORFV2-VEGF was found to be a disulfide-linked homodimer with a subunit of approximately 25 kDa. ORFV2-VEGF showed mitogenic activity on bovine aortic and human microvascular endothelial cells and induced vascular permeability. ORFV2-VEGF was found to bind and induce autophosphorylation of VEGFR-2 and was unable to bind or activate VEGFR-1 and VEGFR-3, but bound the newly identified VEGF165 receptor neuropilin-1. These results indicate that, from a functional viewpoint, ORFV2-VEGF is indeed a member of the VEGF family of molecules, but is unique, however, in that it utilizes only VEGFR-2 and neuropilin-1.

Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L.

A homolog of the vaccinia virus (VAC) interferon resistance gene E3L has been discovered in orf virus strain NZ-2, a parapoxvirus that infects sheep, goats and humans. The gene is located 20 kb from the left terminus of the orf virus genome and is transcribed towards this terminus. RNase protection studies have been used to define the limits of the gene and Northern analysis revealed that it is expressed early in infection. The predicted amino acid sequence of the orf virus protein shares 31% identity (57% similarity) with the VAC E3L protein. Four of the six residues identified as being essential to dsRNA binding in the vaccinia virus protein are conserved in the orf virus protein whilst the other two amino acid changes are conservative substitutions. The orf virus gene has been sequenced in two other orf virus strains which vary markedly in their ability to produce experimental lesions in vivo. Their predicted protein sequences vary by less than 3% from the NZ-2 protein. The recombinant orf virus protein, expressed as a fusion protein in E. coli, bound double-stranded (ds)RNA but not dsDNA, single-stranded (ss)DNA or ssRNA . This is the first demonstration of a VAC E3L-like gene encoded by a parapoxvirus.

Ovine diseases. Orf.

Orf virus is an epitheliotropic DNA parapoxvirus with a worldwide distribution that induces acute pustular lesions in the skin of sheep, goats and man. Genetic mapping and sequencing of the orf virus genome have revealed that orf virus has a typical poxvirus distribution of genes, with those essential for viral DNA synthesis, replication and packaging located in the central region, and those involved in virulence concentrated in the terminal regions. The immune and inflammatory response to orf virus infection in the skin and local lymph is vigorous and typical of an anti-viral response, involving CD4+ helper and CD8+ cytotoxic T cells, interferons and antibodies. In spite of this, the virus can repeatedly infect sheep. Host acquired immunity involving CD4+ T cells and interferons is effective in controlling the extent of viral replication, but does not prevent reinfection. Several virus putative virulence genes have been identified. These are: viral homologues of ovine vascular endothelial growth factor (VEGF); ovine IL-10; vaccinia virus E3L interferon resistance gene; and in addition a viral activity that inhibits the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These may be responsible for rescuing orf virus, at least temporarily, from host immunity and aiding viral replication in epidermal cells.

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein.

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully chracterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.

A homolog of interleukin-10 is encoded by the poxvirus orf virus.

A gene encoding a polypeptide with homology to interleukin-10 (IL-10) has been discovered in the genome of orf virus (OV) strain NZ2, a parapoxvirus that infects sheep, goats, and humans. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%), and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpesvirus (67%). The C-terminal region, comprising two-thirds of the OV protein, is identical to ovine IL-10, which suggests that this gene has been captured from its host sheep during the evolution of OV. The IL-10-like gene is transcribed early. Conditioned medium from COS cells transfected with a eukaryotic expression vector containing the OV IL-10-like gene showed the same biological activity as ovine IL-10 in a murine thymocyte proliferation assay. OV IL-10 is likely to be important in immune evasion by OV, since IL-10 is a multifunctional cytokine that has inhibitory effects on nonspecific immunity and Th1 effector function.

A novel strategy for determining protective antigens of the parapoxvirus, orf virus.

We investigated the feasibility of using vaccinia virus (VAC) recombinants containing large multigene fragments of orf virus DNA to identify protective antigens of orf virus (OV). Sixteen OV strain NZ2 DNA fragments with an average size of 11.4 kb were recombined into VAC strain Lister. Each fragment was mapped relative to OV restriction endonuclease maps but was otherwise uncharacterized. Together the recombinants represent 95% of the OV genome in an overlapping manner. Immunofluorescence showed all 16 constructs expressed products recognized by OV antiserum and radioimmune precipitation with the same antiserum allowed the localization of the major antigens of OV to specific recombinants. These data indicated the approximate genomic locations of the genes encoding the OV major antigens and showed that their expression was authentic rather than resulting from read through from VAC sequences adjacent to the site of recombination. Vaccination of OV-naive sheep with the recombinant library provided protection against a subsequent challenge with virulent OV. These data confirm the feasibility of the proposed strategy.

Location, DNA sequence and transcriptional analysis of the DNA polymerase gene of orf virus.

Degenerate oligonucleotides representing conserved regions of various DNA polymerases hybridized to a region located 26 kb from the left end of the orf virus (OV) strain NZ-2 genome. DNA sequence analysis of this region revealed a 3024 bp open reading frame able to encode a protein with 56 percent amino acid identity to the DNA polymerase of vaccinia virus (VAC) and with significant homology to other DNA polymerases. Early transcripts derived from the open reading frame were detected in RNA purified from OV-infected cells, and 5' ends were mapped to a region 8-19 nt downstream from an A/T-rich sequence that resembles VAC early promoters. Unlike the VAC gene, the OV DNA polymerase makes almost exclusive use of G/C coding options. Attempts to substitute the activity of the OV DNA polymerase for its VAC counterpart were unsuccessful. This may indicate that the OV DNA polymerase is incompatible with VAC accessory proteins.

Gene homology between orf virus and smallpox variola virus.

About 47% identity was observed between the deduced amino acid sequences of a protein encoded by a gene of the parapoxvirus orf virus (OV) strain NZ2 and a 6 kDa protein of unknown function reported to be produced by an open reading frame expressed early after infection by the orthopoxvirus Western Reserve vaccinia virus (VAC); the open reading frame is absent from VAC strain Copenhagen. Examination of sequences reported for variola virus (VAR) strains Bangladesh, India, Congo- 1970, Somalia- 1977 and Garcia- 1966 revealed each encoded a correlate 58 amino acid protein. The open reading frame was not reported in the original analyses of these sequences because a lower limit of 60 amino acids was used to identify potential encoded proteins. Inspection of partial reading frames reported for cowpox virus (CWV) and ectromelia virus (EMV) suggested that these viruses might also code for a correlate of the VAC WR protein. DNA sequencing of cloned fragments of CWV and EMV confirmed that both these orthopoxviruses encode closely related, full length variants of the VAC and VAR open reading frames. The OV homologue is coded in the OV strain NZ2 BamHI-E fragment E2L open reading frame, which we reported is transcribed early postinfection; moreover, analysis of an NZ2 variant showed E2L was absent, indicating that E2L, like the VAC cognate, is nonessential for virus replication in cell culture. The parapoxvirus and orthopoxvirus correlates have about 20% amino acid sequence resemblance to African swine fever virus DNA binding protein p10, suggesting an ancestral relation of genes.

Genomic analysis of a transposition-deletion variant of orf virus reveals a 3.3 kbp region of non-essential DNA.

Restriction endonuclease analysis of the DNA extracted orf virus strain NZ2, which had been serially passaged in primary bovine testis cells, revealed a population of variants that had over-grown the wild-type virus. At least three distinct mutant forms were identified in which the right end of the genome had been duplicated and translocated to the left end, accompanied by deletions of sequences at the left end. Sequencing of a single variant isolated from the heterogeneous population revealed that recombination had occurred between non-homologous sequences. In this case, 6.6 kb of DNA at the left end of the genome had been replaced by 19.3 kb from the right end. The transposition resulted in the deletion at the left end of 3.3 kb of DNA encoding three genes and the terminal sequences of a fourth gene. The three genes completely deleted were a homologue of dUTPase, a gene that encodes a protein containing ankyrin-like repeats and a homologue of the 5K gene of the vaccinia virus WR strain. Experimental inoculation of sheep showed that the genes are also non-essential in vivo, but that the size of the lesion was reduced, compared with that induced by the wild-type, and resolved more rapidly.

The establishment of a genetic map of orf virus reveals a pattern of genomic organization that is highly conserved among divergent poxviruses.

The large differences between the G+C content of the orf virus genome and those of other characterized poxviruses have precluded the use of DNA hybridization to establish a gene map of orf virus. Here we have sequenced the ends of cloned restriction endonuclease fragments of the nZ2 strain of orf virus (OV) and used the translated sequences to search protein data bases. Sequence from 15 points found high-scoring matches to data base entries, including 18 vaccinia virus (VAC) genes. We also present 2 kb of sequence from a region near the right terminus of the OV genome and show that it encodes homologs of VAC genes, F9L and F10L. The data presented here in conjunction with published and as yet unpublished data have allowed the construction of a gene map of OV on which 37 genes have been placed. Thirty-two of these genes have homologs in VAC. Alignment of the OV gene map with that of VAC revealed that each OV gene and its VAC counterpart occurred in the same order and orientation on their respective genomes. The intervals between many of the points of sequence were also found to be strikingly similar. The conserved spacing of genes between OV and VAC within the central 88.2 kb of the 139-kb OV genome is not maintained in the termini where insertion, deletion, and translocation have occurred. Parallels are drawn between the data presented here and related data from swinepox virus and capripox virus.

Parapoxvirus of red deer: evidence for its inclusion as a new member in the genus parapoxvirus.

There are three accepted members of the parapoxvirus genus, orf virus (OV), papular stomatitis virus (PSV), and pseudocowpox virus (PCV). OV is maintained in sheep and goats and PSV and PCV in cattle. Restriction endonuclease profiles of the DNA derived from representatives of these established members of the genus were compared with profiles from a parapoxvirus recently isolated from red deer. In no case did the profile of this latter virus (DPV) resemble those generated from the other parapoxviruses. Southern blot hybridization using total DPV DNA as a probe revealed homology between DPV and the central regions of the genomes of the other parapoxviruses but not to their terminal regions. These results indicate that the genome of DPV is as different from the genomes of the three accepted members of the genus as the latter are from each other and argue for the inclusion of DPV as a new member of the parapoxvirus genus.