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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↗

Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions.

In sheep infected with the parapoxvirus orf virus, primary infection orf skin lesions developed and resolved within 8 weeks. Reinfection lesions were smaller and resolved within 3 weeks. The host response in the skin was characterized by an accumulation of neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, B cells and T19+ gammadelta T cells. The magnitude of this accumulation paralleled orf virus replication in the skin. In situ hybridization was used to detect cells expressing interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) mRNAs in orf skin. Cells expressing IL-4 mRNA were not detected at any time after infection. Cells expressing IFN-gamma mRNA were detected after reinfection but not after primary infection. Cells expressing TNF-alpha mRNA included epidermal cells, vascular endothelium and uncharacterized cells that increased more rapidly in the skin after reinfection compared to primary infection. The results are consistent with a prominent role for IFN-gamma in the host immune response controlling the severity of the disease.

Animals↗

Orf virus immuno-modulation and the host immune response.

Orf virus encodes a range of immuno-modulatory genes that interfere with host anti-virus immune and inflammatory effector mechanisms. The function of these reflects the pathogenesis of orf. The orf virus interferon resistance protein (OVIFNR) and virus IL-10 (vIL-10) inhibit interferon production and activity. In addition the vIL-10 suppresses inflammatory cytokine production by activated macrophages and keratinocytes. The virus GM-CSF inhibitory factor (GIF) is a novel virus protein that binds to and inhibits the biological activity of GM-CSF and IL-2. Together, these immuno-modulators target key effector mechanisms of host anti-virus immunity to allow time for virus replication in epidermal cells.

Animals↗

Cytokines and their inhibitors in orf virus infection.

The epitheliotropic parapoxvirus, orf virus, can repeatedly infect sheep skin. A specific immune response is generated as reinfections induce smaller lesions with quicker resolution times than primary lesions. Cyclosporin-A treatment abrogates this partial immunity. Cytokine mRNAs detected in lesion biopsies include the transcripts for IL-1 beta, IL-3 GM-CSF, TNF-alpha and, less reproducibly, IFN-gamma. CD4+ T-cells predominate in afferent lymph draining the site of infection, and are the major source of GM-CSF and IFN-gamma. IL-1 beta and IL-8 are also detected. The orf virus genome contains a homologue of mammalian vascular endothelial growth factor that may enhance virulence and a vaccinia virus E3L-like gene which may inhibit the anti-viral effect of the interferons. A GM-CSF inhibitory activity has also been discovered and has been 'chased' into a 10 kb DNA segment of the orf virus genome. These studies indicate that orf virus may temporarily avoid host immunity by a combination of acute, rapid infection and replication in the epidermis and by producing virulence factors that inhibit protective proteins of the host immune and inflammatory response.

Animals↗

Immunomodulation by virulence proteins of the parapoxvirus orf virus.

Three orf virus putative virulence proteins are described that exhibit immunomodulatory functions. The OVIFNR gene at the left terminus of the viral genome encodes an interferon resistance protein with homology to the E3L gene of vaccinia virus. OVIFNR functions by preventing a dsRNA-dependent kinase from inhibiting virus and cell protein synthesis as part of the interferon-induced anti-viral state within infected cells. The orf virus orthologue of the ovine interleukin-10 (vIL-10) gene is located at the right terminus of the viral genome. Both vIL-10 and host (ovine) IL-10 function in vitro as inhibitors of pro-inflammatory cytokine production by keratinocytes and macrophages, and both inhibit IFN-gamma production from activated peripheral blood lymphocytes. Both the orf virus vIL-10 and ovine IL-10 stimulate mast cell and thymocyte proliferation. In this respect the orf virus IL-10 differs from Epstein Barr virus IL-10 which does not exhibit cell proliferative activity. Finally, the orf virus GM-CSF inhibitory factor gene (GIF) at the right terminus of the viral genome encodes an inhibitor of GM-CSF that also binds IL-2. Together, these viral proteins are capable of inhibiting key components of the ovine anti-virus immune and inflammatory response.

Animals↗

Immunity and counter-immunity during infection with the parapoxvirus orf virus.

Orf virus is a DNA parapoxvirus that causes orf, an acute debilitating skin disease of sheep, goats and humans. In sheep, a vigorous immune response involving neutrophils, dermal dendritic cells, T cells, B cells and antibody is generated after infection. CD4(+) T cells, IFN-gamma and to a lesser extent CD8(+) T cells are involved in partial protection against infection. In spite of this, orf virus can repeatedly infect sheep albeit with reduced lesion size and time to resolution compared to primary infection. This is due at least in part to the action of virus immuno-modulator proteins that interfere with host immune and inflammatory responses. These include: an interferon resistance protein; a viral orthologue of mammalian IL-10 (vIL-10) that is an anti-inflammatory cytokine; and a novel inhibitor of the cytokines GM-CSF and IL-2 (GIF). The virus also encodes a virulence protein that is an orthologue of mammalian vascular endothelial growth factor. The study of the immuno-modulator proteins provides an insight into disease pathogenesis and important elements of a host protective response. This information will be used to devise a rational disease control strategy.

Adjuvants, Immunologic↗

Characterization of a North American orf virus isolated from a goat with persistent, proliferative dermatitis.

The characterization of an orf virus (OV) isolated from skin lesions of a goat kid with severe, persistent, proliferative dermatitis, and designated orf virus-San Angelo 2000 (OV-SA00) strain, is described. The identity of OV-SA00 was confirmed by a combination of methods, including electron microscopy, amplification of specific fragments of viral DNA by polymerase chain reaction, restriction enzyme analysis of viral DNA and gene sequencing. Restriction endonuclease analyses of viral DNA and the protein profile studied by Western blot revealed differences between OV-SA00 strain and the profiles of other OV strains that have been published. The restriction enzyme profile of OV-SA00 was also different from the orf virus vaccine (OV-V) strain used to vaccinate this kid. Comparison of the nucleotide and deduced amino acid sequences indicated that OV-SA00 is closely related to OV-V strain, the Scottish OV strains orf11 and MRI Scab, and the human OV-CE/Shoe strain and more distant to bovine papular stomatitis virus (BPSV) reference strain and the pseudocowpox virus (PCPV)-MNV/Till strain. These results indicate that OV-SA00 is a strain of OV rather than a different parapoxvirus. Further studies are necessary to determine if the severity of orf-induced lesions in this goat kid was the result of individual host susceptibility factors.

Amino Acid Sequence↗

Perianal orf.

The parapox viral infection orf is usually diagnosed without difficulty when the lesions have the characteristic morphology and there is an appropriate history of contact with sheep. Two cases of orf in a perineal location in young children are presented to illustrate modification of the physical signs by flexural occluded sites. Electron microscopy of scrapings from the lesions established the diagnosis.

Anal Canal↗

Molecular characterization of Brazilian isolates of orf virus.

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.

Animals↗