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.

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.

Sequence and functional analysis of a homolog of interleukin-10 encoded by the parapoxvirus orf virus.

Orf virus is a large DNA virus and is the type species of the Parapoxvirus genus of the family Poxviridae. Orf virus infects the epithelium of sheep and goats and is transmissible to humans. Recently we discovered a gene in orf virus that encodes a polypeptide with remarkable homology to mammalian interleukin (IL-10) and viral encoded IL-10s of herpes viruses. 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 herpes virus (67%). The C-terminal region, comprising two-thirds of the orf virus protein, is identical to ovine IL-10 which suggests that this gene has been captured from its host sheep during the evolution of orf virus. In contrast the N-terminal region shows little homology with cellular IL10s and in this respect resembles other viral IL-10s. IL-10 is a pleiotrophic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. IL-10 is a potent anti-inflammatory cytokine with inhibitory effects on non-specific immunity in particular macrophage function and Thl effector function. Our studies so far, indicate, that the functional activities of orf virus IL-10 are the same as ovine IL-10. Orf virus IL-10 stimulates mouse thymocyte proliferation and inhibits cytokine synthesis in lipopolysaccharide-activated ovine macrophages, peripheral blood monocytes and keratinocytes. Infection of sheep with an IL-10 deletion mutant of orf virus has shown that interferon-gamma levels are higher in tissue infected with the mutant virus than the parent virus. The functional activities of IL-10 and our data on orf virus IL-10 suggest a role in immune evasion.

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.

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.

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.

Sequence and transcriptional analysis of an orf virus gene encoding ankyrin-like repeat sequences.

A 1608 bp region located approximately 5.0 kb from the left end of the orf virus (OV) genome (strain NZ2) was sequenced. The sequence revealed a single open reading frame designated G1L. The predicted amino acid sequence of G1L contained eight ankyrinlike repeat sequences. Transcriptional analysis of G1L showed it was transcribed towards the genome terminus during the early phase of infection. S1 nuclease and primer extension analyses showed that the transcriptional start site of the gene was located a short distance downstream from an A + T-rich sequence similar to a vaccinia virus early promoter.

Sequence and transcriptional analysis of a near-terminal region of the orf virus genome.

A 3605 bp region located approximately 6.6 kb from the left end of the orf virus genome (strain NZ2) was sequenced. The sequence revealed two open reading frames, which we have designated G2L and B1L. The predicted amino acid sequences of G2L and B1L were found to be homologous to the vaccinia virus (VAC) F11L and F12L gene products, respectively, and were found to be arranged on the genome in the same orientation and relative position as their VAC counterparts. Transcriptional analysis of both G2L and B1L showed they were transcribed toward the genome terminus during the early phase of infection. S1 nuclease and primer-extension analyses showed that the transcriptional start sites of both genes were located a short distance downstream from A+T-rich sequences, similar to vac virus early promoters.

Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K.

DNA sequence analysis of a 1.55-kb region located 10 kb from the left end of the orf virus NZ-2 strain (OV NZ2) genome revealed an open reading frame, B2L, encoding a protein with a predicted molecular weight of 41.67 kDa. This protein (p42K) shows 42% amino acid sequence identity to the vaccinia virus (VAC) major envelope antigen p37K. In addition, p42K shows homology to a protein encoded by molluscum contagiosum virus (42.8% identity) and another encoded by fowlpox virus (38.3% identity). These proteins are themselves homologues of the VAC p37K. B2L is actively transcribed after the onset of DNA replication and S1 nuclease analysis mapped the 5' end of the transcript to within the sequence TAAATG. A VAC recombinant capable of expressing the p42K gene was constructed and used as an antigen in radioimmune precipitations and lymphocyte transformation assays. These assays demonstrated that OV p42K is one of a limited number of OV proteins to which sheep mount a strong antibody response and which stimulate lymphocytes derived from draining lymph nodes following a natural infection with OV NZ2.

Homologs of vascular endothelial growth factor are encoded by the poxvirus orf virus.

A gene encoding a polypeptide with homology to mammalian vascular endothelial growth factors (VEGFs) has been discovered in the genome of orf virus (OV), a parapoxvirus that affects sheep and goats and, occasionally, humans. The gene is transcribed abundantly early in infection and is found immediately outside the inverted terminal repeat at the right end of the genome. In the NZ2 strain of OV (OV NZ2), the gene encodes a polypeptide with a molecular size of 14.7 kDa, while in another strain, OV NZ7, there is a variant gene that encodes a polypeptide of 16 kDa. The OV NZ2 and OV NZ7 polypeptides show 22 to 27% and 16 to 23% identity, respectively, to the mammalian VEGFs. The viral polypeptides are only 41.1% identical to each other, and there is little homology between the two genes at the nucleotide level. Another unusual feature of these genes is their G+C content, particularly that of OV NZ7. In a genome that is otherwise 63% G+C, the OV NZ2 gene is 57.2% G+C and the OV NZ7 gene is 39.7% G+C. The OV NZ2 gene, but not the OV NZ7 gene, is homologous to the mammalian VEGF genes at the DNA level, suggesting that the gene has been acquired from a mammalian host and is undergoing genetic drift. The lesions induced in sheep and humans after infection with OV show extensive dermal vascular endothelial proliferation and dilatation, and it is likely that this is a direct effect of the expression of the VEGF-like gene.

Conservation of gene structure and arrangement between vaccinia virus and orf virus.

A 3.3-kb BamHI fragment from the center of the orf virus (OV) NZ2 genome has been sequenced, revealing three major open reading frames (ORFs) with homology to vaccinia virus (VAC) genes. These ORFs have been designated F2L, F3R, and F4R and the proteins they encode were found to be homologous to VAC genes H4L (RNA polymerase-associated protein RAP94), H5R (35-kDa virion envelope antigen) and H6R (topoisomerase), respectively. The OV ORFs are arranged on the genome in an almost identical manner to their VAC counterparts revealing the common evolutionary origin of the two viruses despite the extreme difference in their G+C content. Like its VAC counterpart, F3R was shown to be transcribed early and late during infection. S1 and primer extension analysis located the 5' ends of F3R early transcripts to a position 15-16 nt and 5-10 nt, respectively, downstream from an AT-rich sequence resembling a VAC early promoter. The 5' ends of F3R late transcripts were located to an A within the sequence 5'-TAAAG, 41 nt downstream from the early promoter and 17 nt upstream from the initiation codon. S1 analysis of F2L, which is transcribed only late in infection, revealed transcripts initiating from within the sequence 5'-TAAATG. No transcriptional start point could be detected for F4R but the VAC late transcriptional initiation sequence TAAAT was found close to the predicted translational start point. Another late promoter-like sequence, 5'-TAAATG, was found at the 3' end of F2L. This preceded a short ORF tentatively designated as F1L and predicted to be the beginning of a homologue of VAC H3L.

In vivo recognition of orf virus early transcriptional promoters in a vaccinia virus recombinant.

The 4.4-kb BamHI-E fragment of the orf virus (OV) genome contains three discrete open reading frames designated ORF-pp, ORF-1, and ORF-3, all of which are flanked by vaccinia virus-like early transcriptional control sequences. To determine whether the vaccinia transcriptional machinery would recognize these promoters and faithfully transcribe OV genes in vivo the BamHI-E fragment was inserted into the thymidine kinase (TK) locus of vaccinia virus and the recombinant used in transcription studies. Northern blotting analysis of early RNA isolated from 143B-TK- cells infected with the recombinant virus showed that OV genes were transcribed and that the three transcripts of 0.70-(ORF-pp), 0.48- (ORF1), and 0.75-kb (ORF-3) were the same size as their counterparts in OV-infected cells. Analysis of the 5' end of transcripts by S1 nuclease and primer extension showed that the transcriptional start points (tsp) of ORF-pp, ORF-1, and ORF-3 in the recombinant were identical or within four nucleotides of the tsps of the same ORFs in OV. However, there were quantitative differences. ORF-1 was transcribed more efficiently in recombinant virus-infected cells than in those infected with OV and analysis of the putative promoter, 5'-AAAATTGTAAATGTA, showed that it was similar to the 7.5-kDa early promoter of vaccinia virus. This demonstrates that the transcriptional control sequences of OV genes are recognized by vaccinia virus transcriptional factors but that quantitative differences exist suggesting that the generically different transcriptional factors have different promoter sequence requirements for maximal transcription.

In vitro recognition of an orf virus early promoter in a vaccinia virus extract.

DNA fragments containing varying lengths of the 5' end of an orf virus early gene (ORF3) and its associated promoter were introduced into sodium deoxycholate-solubilized vaccinia virus extracts capable of initiating transcription in vitro from vaccinia virus early promoters. After separation of the radiolabelled products of the reactions on a 5% polyacrylamide/7 M urea gel, discrete transcripts were detected the sizes of which were consistent with initiation of transcription from the orf virus early promoter. This is the first demonstration in a functional assay of the conservation of early transcriptional promoters between an orthopoxvirus and a parapoxvirus.

Vaccinia virus-like early transcriptional control sequences flank an early gene in orf virus.

The purpose of this study was to map the initiation (tsp) and termination points of transcripts arising from an open reading frame (ORF3) found in the inverted terminal repeat of the orf virus genome and also, to identify probable transcriptional control sequences. Early transcripts of approx. 0.76 kb were mapped to ORF3 and found to be transcribed toward the ends of the genome. Using the S1 nuclease and primer-extension methods, the bulk of the tsp were mapped to a position 12-13 nucleotides (nt) downstream from a sequence which resembles A + T-rich vaccinia virus early promoters. The 5' ends were 81-82 nt upstream from the first ATG in ORF3. Most of 3' ends of the transcripts mapped to a region 24-32 nt downstream from a T5NT sequence found near the ORF3 stop codon. A second transcription termination point was found 25 nt downstream from another T5NT sequence located downstream and separated by 85 nt from the first. These results infer that the A + T-rich, early transcriptional control sequences found in other poxvirus genomes have been conserved in the G + C-rich genome of orf virus.

Comparison of a DNA-DNA dot-blot hybridisation assay with light microscopy and radioimmunoassay for the detection of a nuclear polyhedrosis virus.

A dot-blot hybridisation assay was developed for the detection of a nuclear polyhedrosis virus (NPV) and was compared to light microscopy and radioimmunoassay (RIA). Using cloned NPV DNA labelled with 32P as a probe, a number of hybridisation assay procedures was examined. The assay was found to be more sensitive than differential staining, phase-contrast microscopy, or indirect solid-phase RIA with as few as 20 occlusion bodies (150 pg DNA) being detected. Samples do not require prior purification or DNA extraction. The assay was shown to be specific for NPV and has the potential to detect and discriminate between strains of the virus. With little modification the assay may be used to detect other insect viruses.