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Parapoxvirus papillomatosis in the muskoxen (Ovibos moschatus): genetical differences between the virus causing new outbreak in a vaccinated herd, the vaccine virus and a local orf virus.

Since 1981 a domesticated muskoxen herd had been successfully vaccinated against papillomatosis with homogenated, glutaraldehyde inactivated papilloma tissue. In the fall of 1985 a new clinical outbreak of disease occurred, affecting previously infected as well as vaccinated animals. The purification of parapox virions directly from papilloma tissue and orf scabs collected in a local sheep farm was followed by restriction endonuclease analysis of viral DNA. The morphological identity of purified virus was controlled by electron microscopy. Comparison of restriction endonuclease digests (10 different enzymes) by gel electrophoresis demonstrated that the muskoxen parapoxvirus from the new outbreak 1985 differed considerably from the 2 other isolates (muskoxen 1981 and local orf). The latter viruses demonstrated a high degree of homology, but differences were evident after digestion with the enzyme EcoRI. During metrizamide gradient purification minor bands containing morphologically intact virions were isolated in addition to the major fractions. The restriction enzyme digests indicated that the virions of the minor bands differed from those in the major bands.

Animals↗

Polymerase chain reaction for laboratory diagnosis of orf virus infections.

BACKGROUND: The orf virus of sheep and goats is one of several zoonotic parapoxviruses. In the ovine/caprine host it causes contagious ecthyma (contagious pustular dermatitis, scabby mouth), but in humans it normally causes solitary or clustered orf lesions, typically on hands, arms or face. In addition to disease in the animals, the virus can be quite a nuisance as an occupational hazard in farmers and butchers. Clinical diagnosis is often possible, but laboratory diagnosis is sometimes necessary. For virus isolation, primary ovine or bovine cells, not routinely present, are needed. Serological methods exist, but electron microscopy is the most commonly used method. OBJECTIVES: To develop a reliable method for the laboratory diagnosis of orf zoonoses, without virus culture and without access to an electron microscope. STUDY DESIGN: A suitable primer pair was designed for orf polymerase chain reaction (PCR), using the Oligo software and sequence information from GenBank. Orf positive controls and specimens were kindly provided by several public health centers. Molluscum contagiosum specimens were provided by a dermatologist. HSV-1, HSV-2 and VZV positive swab specimens came from our routine diagnostic service. Asymptomatic skin specimens were obtained from sheep heads from the abattoir, and swab specimens from the heads of asymptomatic sheep. Selected amplified orf PCR positive specimens were sequenced to ensure the authenticity of the PCR products. Orf positive specimens were sent to another laboratory for electron microscopy. RESULTS AND CONCLUSIONS: A robust PCR was developed, with very small inter-run variation. All specificity demands were met, and the sensitivity seems to be good or excellent. All negative specificity controls from cell cultures and non-orf viruses were negative. Twenty-two (95.7%) of 23 scab or swab specimens with suspected orf etiology were orf PCR positive. Five of eight skin specimens from sheep heads from the abattoir were positive, and all 11 swab specimens from asymptomatic sheep were negative. Electron microscopy demonstrated orf-like particles in orf-PCR positive specimens. This PCR seems to be suitable as a diagnostic test for orf in humans, but asymptomatic virus shedding in sheep or goats may complicate veterinary applications of the assay.

Animals↗

Phylogenetic correlation of Greek and Italian orf virus isolates based on VIR gene.

Thirteen orf virus isolates obtained during the time period between 1995 and 2004 from crusted scab lesions of nine sheep and four goats from different geographical areas of Greece and Italy with suspected contagious ecthyma infection were analyzed. DNA of all isolates was successfully amplified by PCR with the primers 045F-045R and identified them as parapox virus. Partial DNA sequence of orf virus interferon resistant (VIR) gene, phylogenetic analysis of the available isolates and amino acid comparison of the interferon resistance protein encoded by this genomic region was carried out. According to the results of the present report a precise characterisation of the genomic region studied might provide evidence for the genetic variation and movement of the circulating orf virus strains.

Amino Acid Sequence↗

Comparative sequence analysis of major envelope protein gene (B2L) of Indian orf viruses isolated from sheep and goats.

Orf virus (ORFV), the type species of Parapoxvirus, is responsible for contagious ecthyma in sheep and goats. In the present report, sequence analysis of major envelope gene (B2L) of four Indian orf virus isolates originating two each from sheep and goats was carried out. These recent isolates belonged to different outbreaks that occurred in Kumaon hills and adjoining plains during 2004-2005. Preliminary screening of the scab samples was carried out by diagnostic PCR. Full-length B2L gene encoding for immunogenic major envelope protein from all the four ORFV isolates was amplified by PCR and the amplicons (1206 bp) were cloned and sequenced. Comparative sequence analysis revealed an open reading frame of 1137 nucleotides (nt) encoding a polypeptide of 378 amino acids (aa). Indian isolates were highly related amongst themselves with sequence identity of over 97% at the nt and aa level. Further, they showed 97-98% sequence identity with sequences of other ORFV isolates from around the world; while 94-95 and 82.7-83.8% sequence identity was observed, respectively, with pseudocowpox and bovine papular stomatitis viruses--the other members of the genus. Phylogenetic analysis also showed that these Parapoxviruses from sheep and goats are closely related to other orf viruses reported worldwide.

Amino Acid Sequence↗

A comparison of the anti-inflammatory and immuno-stimulatory activities of orf virus and ovine interleukin-10.

Orf virus causes pustular skin lesions (orf) in sheep, goats and humans. The virus encodes an interleukin-10 (orfvIL-10) that is identical in amino acid composition to ovine IL-10 (ovIL-10) over the C terminal two-thirds of the polypeptide, but not in the N terminal third. The immuno-suppressive and immuno-stimulatory activities of orfvIL-10 and ovIL-10 were compared. Both orfvIL-10 and ovIL-10 inhibited TNF-alpha and IL-8 cytokine production from stimulated ovine macrophages and keratinocytes and IFN-gamma and GM-CSF production from peripheral blood lymphocytes. OrfvIL-10 and ovIL-10 co-stimulated both ovine and murine mast cell proliferation in conjunction with IL-3 (ovine) or IL-4 (murine). Isoleucine at position 87 (Ile(87)) of the mature human IL-10 (huIL-10) has been reported as essential for the immuno-stimulatory activity of huIL-10. In spite of the differences in amino acids within the N-terminal third of orfvIL-10 compared with ovIL-10 and substitution of Ile(87) with Ala(87) in ovIL-10, these variants of ovIL-10 and orfvIL-10 all co-stimulated mast cell proliferation and inhibited macrophage IL-8 production. As ovIL-10 and orfvIL-10 have a similar structure to huIL-10 and conserved receptor-binding residues, it was concluded that Ile(87) is not essential for IL-10 immuno-stimulatory activity. Finally, ovine keratinocytes do not express ovIL-10. This might explain why orf virus has evolved a viral IL-10.

Amino Acid Sequence↗

Comparative analysis of genome sequences of three isolates of Orf virus reveals unexpected sequence variation.

Orf virus (ORFV) is the type species of the Parapoxvirus genus. Here, we present the genomic sequence of the most well studied ORFV isolate, strain NZ2. The NZ2 genome is 138 kbp and contains 132 putative genes, 88 of which are present in all analyzed chordopoxviruses. Comparison of the NZ2 genome with the genomes of 2 other fully sequenced isolates of ORFV revealed that all 3 genomes carry each of the 132 genes, but there are substantial sequence variations between isolates in a significant number of genes, including 9 with inter-isolate amino acid sequence identity of only 38-79%. Each genome has an average of 64% G+C but each has a distinctive pattern of substantial deviation from the average within particular regions of the genome. The same pattern of variation was also seen in the genome of another parapoxvirus species and was clearly unlike the uniform patterns of G+C content seen in all other genera of chordopoxviruses. The availability of genomic sequences of three orf virus isolates allowed us to more accurately assess likely coding regions and thereby revise published data for 24 genes and to predict two previously unrecognized genes.

Base Composition↗

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.

Amino Acid Sequence↗

Analysis of genomic rearrangement and subsequent gene deletion of the attenuated Orf virus strain D1701.

The orf virus (OV) strain D1701 belongs to the genetically heterogenous parapoxvirus (PPV) genus of the family Poxviridae. The attenuated OV D1701 has been licensed as a live vaccine against contagious ecthyma in sheep. Detailed knowledge on the genetic structure and organization of this PPV vaccine strain is an important prerequisite to reveal possible genetic mechanisms of PPV attenuation. The present study demonstrates a genomic map of the approximately 158 kbp DNA of OV D1701 established by hybridization studies of cloned restriction fragments covering the complete viral genome. The results show an enlargement of the inverted terminal repeats (ITR) to up to 18 kbp due to recombination between nonhomologous sequences during cell culture adaptation. DNA sequencing of the region adjacent to the ITR junction revealed the absence of one open reading frame designated E2L. In contrast to a transposition-deletion variant of the New Zealand OV strain NZ2 (Fleming et al., 1995) the two genes E3L (a homologue of dUTPase) and G1L neighbouring E2L are retained in OV D1701. DNA and RNA analyses proved the presence of E2L gene in wild-type OV isolated directly from scab material. The data presented indicate that the E2L gene is nonessential for virus replication in vitro and in vivo, and may represent one important viral gene in determining virulence and pathogenesis of OV.

Amino Acid Sequence↗

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.

Amino Acid Sequence↗

Natural transmission of orf virus from clinically normal ewes to orf-naive sheep.

The apparent natural transmission of orf virus from clinically normal ewes to susceptible sheep was observed during a border disease vaccine experiment. The 14 susceptible sheep were persistently infected with border disease virus and had been reared indoors in isolation from other sheep since birth. Their ages ranged from two to four years and they were housed in two groups; group 1 consisted of four sheep persistently infected with the Moredun strain of border disease virus and group 2 consisted of 10 sheep persistently infected with the Oban strain of the virus. On day 0, six sheep were removed from group 2 and rehoused. To the remaining four sheep in each group were added eight four- to six-year-old pregnant conventionally reared ewes at 48 days gestation. Fourteen days later the four sheep in group 1 were moved to another pen housing eight similar five-year-old pregnant ewes at 48 days' gestation, and the four sheep from group 2 were rehoused with their original stallmates. Twenty-one days later lip lesions typical of orf were first observed on the sheep from both groups and the disease spread to all the sheep persistently infected with border disease virus over the next four weeks. Virological and serological evidence demonstrated that the source of infection for the sheep was almost certainly the conventionally reared ewes, on which no lesions resembling orf were observed at any time during the study.

Animals↗

A homologue of retroviral pseudoproteases in the parapoxvirus, orf virus.

The nucleotide sequence of a near-terminal region of orf virus DNA was determined. Examination of the sequence revealed an open reading frame encoding a peptide with significant amino acid homology to the pseudoprotease domains recently identified in a number of retroviruses including mouse mammary tumor virus, simian Mason-Pfizer virus, maedi-visna virus, and equine infectious anaemia virus. The orf virus pseudoprotease shares up to 28% amino acid homology with retroviral pseudoproteases and appears to be a discrete transcriptional unit rather than a subunit of a larger polypeptide as is the case in retroviruses. The sharing of amino acid composition across such wide taxonomic boundaries suggests that this polypeptide has a functional significance in both retroviruses and poxviruses.

Animals↗

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.

Amino Acid Sequence↗

Genomes of the parapoxviruses ORF virus and bovine papular stomatitis virus.

Bovine papular stomatitis virus (BPSV) and orf virus (ORFV), members of the genus Parapoxvirus of the Poxviridae, are etiologic agents of worldwide diseases affecting cattle and small ruminants, respectively. Here we report the genomic sequences and comparative analysis of BPSV strain BV-AR02 and ORFV strains OV-SA00, isolated from a goat, and OV-IA82, isolated from a sheep. Parapoxvirus (PPV) BV-AR02, OV-SA00, and OV-IA82 genomes range in size from 134 to 139 kbp, with an average nucleotide composition of 64% G+C. BPSV and ORFV genomes contain 131 and 130 putative genes, respectively, and share colinearity over 127 genes, 88 of which are conserved in all characterized chordopoxviruses. BPSV and ORFV contain 15 and 16 open reading frames (ORFs), respectively, which lack similarity to other poxvirus or cellular proteins. All genes with putative roles in pathogenesis, including a vascular endothelial growth factor (VEGF)-like gene, are present in both viruses; however, BPSV contains two extra ankyrin repeat genes absent in ORFV. Interspecies sequence variability is observed in all functional classes of genes but is highest in putative virulence/host range genes, including genes unique to PPV. At the amino acid level, OV-SA00 is 94% identical to OV-IA82 and 71% identical to BV-AR02. Notably, ORFV 006/132, 103, 109, 110, and 116 genes (VEGF, homologues of vaccinia virus A26L, A33R, and A34R, and a novel PPV ORF) show an unusual degree of intraspecies variability. These genomic differences are consistent with the classification of BPSV and ORFV as two PPV species. Compared to other mammalian chordopoxviruses, PPV shares unique genomic features with molluscum contagiosum virus, including a G+C-rich nucleotide composition, three orthologous genes, and a paucity of nucleotide metabolism genes. Together, these data provide a comparative view of PPV genomics.

Animals↗

In vivo T-cell subset depletion suggests that CD4+ T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep.

In vivo lymphocyte subset depletion offers a unique opportunity to study the roles of different cellular components of the immune system of sheep during infection with orf virus. Lambs were depleted of specific lymphocyte subsets by the intravenous administration of monoclonal antibodies against ovine lymphocyte surface markers and then challenged with orf virus. The skin lesions that developed were scored visually as to their severity. Blood samples were collected to monitor the lymphocyte depletions and to measure orf-virus-specific antibody levels. Skin biopsies were collected from the lesion site and studied to determine the course of the infection and the presence of various cell types and orf virus. All the sheep developed orf virus lesions after infection. All three of the CD4-depleted lambs were unable to clear virus from their skin and did not have an antibody response to the virus. Virus was also detected in the skin of one each of the three CD8-depleted, WC1-depleted and control sheep on the final day of the trial. CD8(+) lymphocytes did not appear to be essential for viral clearance later in the infection. Depletion of the majority of gammadelta(+) T-cells did not affect the outcome of orf virus infection. In sheep with high orf-virus-specific antibody titres at the time of infection, orf lesions healed faster than lesions in sheep with low antibody levels, and this occurred regardless of the lymphocyte depletion status of the animals. This study suggests that the presence of CD4(+) T-cells and orf-virus-specific antibodies are important for the control of viral replication in the skin of infected sheep.

Animals↗

Activities of acyclic nucleoside phosphonates against Orf virus in human and ovine cell monolayers and organotypic ovine raft cultures.

Orf virus, a member of the Parapoxvirus genus, causes a contagious pustular dermatitis in sheep, goats, and humans. Previous studies have demonstrated the activity of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; cidofovir; Vistide) against orf virus in cell culture and humans. We have evaluated a broad range of acyclic nucleoside phosphonates (ANPs) against several orf virus strains in primary lamb keratinocytes (PLKs) and human embryonic lung (HEL) monolayers. HPMPC, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6- diaminopurine (HPMPDAP), and (R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) were three of the most active compounds that were subsequently tested in a virus yield assay with PLK and HEL cells by virus titration and DNA quantification. HPMPC, HPMPDAP, and HPMPO-DAPy were evaluated for their activities against orf virus replication in organotypic epithelial raft cultures from differentiated PLK cells. At the highest concentrations (50 and 20 microg/ml), full protection was provided by the three drugs, while at 5 microg/ml, only HPMPDAP and HPMPC offered partial protection. The activities of the three compounds in the raft culture system were confirmed by quantification of infectious virus and viral DNA. These findings provide a rationale for the use of HPMPC and other ANPs in the treatment of orf (contagious ecthyma) in humans and animals.

Adenine↗

The genome of orf virus: restriction endonuclease analysis of viral DNA isolated from lesions of orf in sheep.

The purification of orf virus directly from scab material from clinical cases of orf in sheep and restriction endonuclease analysis of the viral DNA is described. Between 7 x 10(9) and 1.6 x 10(11) virus particles, and 0.7 to 22.8 micrograms of viral DNA could be recovered from 1g of scab material. Considerable heterogeneity was observed between different field isolates when restriction endonuclease digests of orf DNA were compared by gel electrophoresis. It was also shown, for two isolates, that these fragment patterns did not change after plaque purification and passage in cell culture. It is suggested that restriction endonuclease analysis of viral DNA offers a convenient method of identification of isolates of orf virus. The molecular weight of orf DNA was determined and found to be 88.8 x 10(6).

Animals↗

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.

Amino Acid Sequence↗

[Recent information about the orf virus of small ruminants].

New scientific findings in the field of immunobiology and diagnosis of parapoxvirus ovis (Orf-virus) as the causal agent of a zoonosis are presented. The adaptation of Orf-virus to cell lines and its in vitro multiplication without difficulties offer the possibility for extensive studies into the biology of parapoxviruses. The development of monoclonal antibodies (MAB) against an attenuated Orf-virus strain (D-1701) led to the elaboration of a simplified, cheap and highly sensitive "antigen detection ELISA" as a diagnostic tool. Moreover the MAB's can be used to identify parapoxvirus in cells of infected tissues (indirect immuno-peroxidase technique) which is an important precondition for more detailed surveillance of the pathogenesis of parapoxvirus infections. The immunobiological behavior of the Orf-virus in its natural hosts (small ruminants) as well as in non-susceptible species (e.g. laboratory animals) is explained by the course of cellular and humoral immune reactions following infection or vaccination. The problems of combat measures against contagious ecthyma (scabby mouth) of sheep and the human Orf-virus infections are discussed.

Animals↗