David M. Haig

Malignant catarrhal fever: a review.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle and other ungulates caused by the ruminant gamma-herpesviruses alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). These viruses cause inapparent infection in their reservoir hosts (wildebeest for AlHV-1 and sheep for OvHV-2), but fatal lymphoproliferative disease when they infect MCF-susceptible hosts, including cattle, deer, bison, water buffalo and pigs. MCF is an important disease wherever reservoir and MCF-susceptible species mix and currently is a particular problem in Bali cattle in Indonesia, bison in the USA and in pastoralist cattle herds in Eastern and Southern Africa. MCF is characterised by the accumulation of lymphocytes (predominantly CD8(+) T lymphocytes) in a variety of organs, often associated with tissue necrosis. Only a small proportion of these lymphocytes appear to contain virus, although recent results with virus gene-specific probes indicate that more infected cells may be present than previously thought. The tissue damage in MCF is hypothesised to be caused by the indiscriminate activity of MHC-unrestricted cytotoxic T/natural killer cells. The pathogenesis of MCF and the virus life cycle are poorly understood and, currently, there is no effective disease control. Recent sequencing of the OvHV-2 genome and construction of an AlHV-1 bacterial artificial chromosome (BAC) are facilitating studies to understand the pathogenesis of this extraordinary disease. Furthermore, new and improved methods of disease diagnosis have been developed and promising vaccine strategies are being tested. The next few years are likely to be exciting and productive for MCF research.

Orf Virus Encodes a Novel Secreted Protein Inhibitor of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2

ABSTRACT The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a Kd of 369 pM and ovine IL-2 with a Kd of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

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.

The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon-inducible, double-stranded RNA-dependent kinase.

The parapoxvirus orf virus was resistant to type 1 (IFN‐α) and type 2 (IFN‐γ) interferons in cultures of ovine cells. The recently identified orf virus OV20.0L gene exhibits 31% predicted amino acid identity to the vaccinia virus E3L interferon‐resistance gene, and is referred to as the (putative) orf virus interferon‐resistance gene (OVIFNR). The objective of this study was to determine whether OVIFNR was involved in interferon resistance. Recombinant OVIFNR as a thioredoxin fusion protein (OVIFNR–Tx) inhibited the activation (by autophosphorylation) of an interferon‐inducible, double‐stranded (ds) RNA‐dependent kinase (PKR) of sheep, which was shown to bind dsRNA (poly I:C). PKR in other species is involved in the inhibition of protein synthesis as part of the antiviral state in infected cells. Virus‐infected cell lysates, but not control lysates, from cells grown in the presence of cytosine arabinoside also contained PKR inhibitory activity, which indicated that the inhibitory activity was associated with early viral gene expression. Significantly, the OVIFNR gene expressed in interferon‐treated ovine fibroblasts protected the unrelated Semliki Forest virus from the antiviral effect of both type 1 and type 2 interferons. Taken together, the results indicate that the OVIFNR gene functions as an interferon‐resistance gene, the product of which inhibits PKR in a similar way to the vaccinia virus E3L gene product.

Molecular evolution of bovine Toll-like receptor 2 suggests substitutions of functional relevance

BackgroundThere is accumulating evidence that polymorphism in Toll-like receptor (TLR) genes might be associated with disease resistance or susceptibility traits in livestock. Polymorphic sites affecting TLR function should exhibit signatures of positive selection, identified as a high ratio of non-synonymous to synonymous nucleotide substitutions (ω). Phylogeny based models of codon substitution based on estimates of ω for each amino acid position can therefore offer a valuable tool to predict sites of functional relevance. We have used this approach to identify such polymorphic sites within the bovine TLR2 genes from ten Bos indicus and Bos taurus cattle breeds. By analysing TLR2 gene phylogeny in a set of mammalian species and a subset of ruminant species we have estimated the selective pressure on individual sites and domains and identified polymorphisms at sites of putative functional importance.ResultsThe ω were highest in the mammalian TLR2 domains thought to be responsible for ligand binding and lowest in regions responsible for heterodimerisation with other TLR-related molecules. Several positively-selected sites were detected in or around ligand-binding domains. However a comparison of the ruminant subset of TLR2 sequences with the whole mammalian set of sequences revealed that there has been less selective pressure among ruminants than in mammals as a whole. This suggests that there have been functional changes during ruminant evolution. Twenty newly-discovered non-synonymous polymorphic sites were identified in cattle. Three of them were localised at positions shaped by positive selection in the ruminant dataset (Leu227Phe, His305Pro, His326Gln) and in domains involved in the recognition of ligands. His326Gln is of particular interest as it consists of an exchange of differentially-charged amino acids at a position which has previously been shown to be crucial for ligand binding in human TLR2.ConclusionWithin bovine TLR2, polymorphisms at amino acid positions 227, 305 and 326 map to functionally important sites of TLR2 and should be considered as candidate SNPs for immune related traits in cattle. A final proof of their functional relevance requires further studies to determine their functional effect on the immune response after stimulation with relevant ligands and/or their association with immune related traits in animals.

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 IL-10s and in this respect resemble 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 Th1 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-γ 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.

Cloning and expression of a cDNA encoding ovine granulocyte-macrophage colony-stimulating factor.

A cDNA encoding ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been cloned using the polymerase chain reaction. The nucleotide sequence is approx. 93% identical to the published bovine GM-CSF-encoding sequence, 84% to the human sequence and 73% to the murine sequence. The deduced amino acid sequence of the ovine GM-CSF protein was found to be 80% identical to both the human and bovine proteins and 57% to the murine protein. Transient expression of recombinant ovine GM-CSF in COS-1 cells was obtained and its biological activity investigated in a bone-marrow colony-forming assay. Ovine GM-CSF was found to promote the formation of granulocyte-macrophage colonies as well as eosinophil, neutrophil and monocyte/macrophage colonies, an activity characteristic of GM-CSF in other species. Recombinant human GM-CSF was found to have no proliferative effect on ovine bone-marrow cells.

Orf virus-encoded interleukin-10 stimulates the proliferation of murine mast cells and inhibits cytokine synthesis in murine peritoneal macrophages

Orf virus (ORFV) is the type species of the parapoxvirus genus and produces cutaneous pustular lesions in sheep, goats and humans. The genome encodes a polypeptide with remarkable homology to interleukin-10 (IL-10), particularly ovine IL-10, and also to IL-10-like proteins encoded by Epstein-Barr virus (EBV) and equine herpesvirus. IL-10 is a pleiotropic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. We have expressed and purified C-terminal FLAG and His(6)-tagged versions of ORFV-IL-10 and shown that ORFV-IL-10 costimulates murine mast cells (MC/9) and inhibits tumour necrosis factor-alpha synthesis in activated mouse peritoneal macrophages. Our results demonstrate that although ORFV-IL-10 is structurally similar to EBV-IL-10 it has evolved a different spectrum of activities. EBV-IL-10 does not stimulate the proliferation of thymocytes or mast cells whereas ORFV-IL-10 has both of these activities. Recent studies show that the critical difference in molecular structure of human IL-10 and EBV-IL-10, which may be the basis of their functional differences, is linked to a single amino acid substitution. Consistent with the activity spectrum reported here for ORFV-IL-10, the viral gene encodes the critical amino acid seen in human IL-10. Although the ORFV-IL-10 gene has clearly undergone significant evolutionary change at the nucleotide level compared with ovine IL-10, it has largely retained the polypeptide structure and functional characteristics of its ovine counterpart, suggesting that mutations of the gene to a potentially more potent immunosuppressive form may compromise the co-existence of host and virus.

Orf virus infection and host immunity.

Purpose of review A summary of recent advances in our knowledge of the biology of orf virus is presented to illustrate the interaction of a zoonotic pathogen with host skin. This is providing novel and interesting data on the viral mechanism of skin infection and the host response. Recent findings The full genome sequences of two parapoxviruses (orf virus and bovine papular stomatitis virus) have recently been published, defining the parapoxvirus genus at the molecular genetic level. This, along with more detailed characterization of viral immuno-modulatory proteins, is providing an insight into the acquisition of host genes and the mechanism of pathogenesis. A new chemokine-binding protein has been discovered with unique features. Structure–function analysis of the viral granulocyte/macrophage colony-stimulating factor inhibitory factor has revealed a similarity to type 1 cytokine receptors. The viral vascular endothelial growth factor-E stimulates angiogenesis in the skin without the side effects seen with cellular vascular endothelial growth factor-A, and may have therapeutic potential. Finally, orf virus is proving useful both as an immuno-modulator and as a vector for the expression of foreign antigens in non-permissive species. Summary Orf virus infection provokes a vigorous skin immune response. However, the virus has acquired a range of immuno-modulatory/pathogenesis-related genes that function to limit (at least transiently) the effectiveness of host immunity. With the advent of the orf virus genome sequence, the study of this dynamic process will provide important insights into virus pathogenesis and the host skin immune response to infection.

Constitutive activation of Lck and Fyn tyrosine kinases in large granular lymphocytes infected with the γ‐herpesvirus agents of malignant catarrhal fever

Large granular lymphocytes (LGL) with a T or natural killer (NK) lymphoblast morphology and indiscriminate (non‐major histocompatibility complex‐linked) cytotoxicity for a variety of target cells can be derived in culture from the tissues of animals infected with either alcelaphine herpesvirus‐1 (AlHV‐1) or ovine herpesvirus‐2 (OvHV‐2). In this study, LGL survival in the absence of exogenous interleukin‐2 was inhibited by the protein kinase inhibitor genestein, but not the p70 s6 kinase inhibitor rapamycin. Constitutive activation of the src kinases Lck and Fyn was demonstrated in a bovine LGL line infected with OvHV‐2 and in two rabbit LGL lines infected with AlHV‐1. The p44 erk1 and p42 erk2 mitogen‐activated protein kinases (MAPK) were also constitutively activated in the LGLs but not control T cells. Lck and Fyn kinase activity in the LGLs did not increase after mitogen (concanavalin A or concanavalin A plus phorbol ester) stimulation of the cells, in contrast to control T cells. Control T cells, but not the LGLs, proliferated after mitogen stimulation. An analysis of tyrosine phosphorylated proteins in the cells indicated that the LGLs exhibited some similarities and differences to activated control T cells. The results demonstrate that the activated phenotype of the LGLs, associated with malignant catarrhal fever virus infection and in the absence of exogenous interleukin‐2, involves constitutively activated Lck and Fyn kinases. These are normally crucial for the initial activation of T cells via several cell‐surface receptors (e.g. the T‐cell receptor and CD2). The inability of the LGLs to proliferate in response to mitogen may be due to an inability of Lck and Fyn to become further activated after mitogen stimulation.