A field vaccine trial in Tanzania demonstrates partial protection against malignant catarrhal fever in cattle.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle that, in East Africa, results from transmission of the causative virus, alcelaphine herpesvirus 1 (AlHV-1), from wildebeest. A vaccine field trial involving an attenuated AlHV-1 virus vaccine was performed over two wildebeest calving seasons on the Simanjiro Plain of northern Tanzania. Each of the two phases of the field trial consisted of groups of 50 vaccinated and unvaccinated cattle, which were subsequently exposed to AlHV-1 challenge by herding toward wildebeest. Vaccination resulted in the induction of virus-specific and virus-neutralizing antibodies. Some cattle in the unvaccinated groups also developed virus-specific antibody responses but only after the start of the challenge phase of the trial. PCR of DNA from blood samples detected AlHV-1 infection in both groups of cattle but the frequency of infection was significantly lower in the vaccinated groups. Some infected animals showed clinical signs suggestive of MCF but few animals went on to develop fatal MCF, with similar numbers in vaccinated and unvaccinated groups. This study demonstrated a baseline level of MCF-seropositivity among cattle in northern Tanzania of 1% and showed that AlHV-1 virus-neutralizing antibodies could be induced in Tanzanian zebu shorthorn cross cattle by our attenuated vaccine, a correlate of protection in previous experimental trials. The vaccine reduced infection rates by 56% in cattle exposed to wildebeest but protection from fatal MCF could not be determined due to the low number of fatal cases.

Toll-like receptor gene expression in fresh and archived ovine pseudoafferent lymph DEC205+ dendritic cells.

Toll-like receptors (TLRs) are key regulators of the innate and adaptive immune response to bacterial, viral and fungal pathogens. To date, 10 human TLRs and 13 mouse TLRs have been identified and they exhibit tissue-specific mRNA/protein expression patterns. We recently cloned and characterized 10 ovine TLR genes. The present study was carried out to determine the expression profile of TLRs 1-10 in fresh and archived ovine pseudoafferent lymph (pAL) cells and pAL dendritic cells (pALDCs) using two-step quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with ovine specific primer/probe sets. Dendritic cells are important in the initiation and maintenance of immune responses and express a spectrum of pattern-recognition receptors (that includes the TLRs). Fresh and archived total pAL cells expressed all 10 ovine TLRs to a broadly similar extent and TLR1-10 mRNA expression was observed in DEC205(hi) pALDCs. In addition, there were changes in particular TLR transcript levels in DEC205(hi) pALDC in archived lymph samples at two time points after orf virus reinfection. The results show that frozen archived cells can be used for retrospective TLR gene expression analysis. Furthermore, changes in TLR gene expression in DEC205(hi) pALDC after orf virus reinfection in the skin of sheep suggests that more detailed analyses of TLR gene expression changes during disease processes are worthwhile. These data will be useful to inform future studies on the role of TLRs in disease pathogenesis and control.

Production and utilization of interleukin-15 in malignant catarrhal fever.

Malignant catarrhal fever (MCF) is an often fatal lymphoproliferative disease of ungulates caused by either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). The pathogenesis of MCF is poorly understood, but appears to involve an auto-destructive pathology whereby cytotoxic lymphocytes destroy areas of a variety of tissues. The cytokine interleukin-15 (IL-15) is involved in the development and maintenance of cytotoxic lymphocytes and may therefore have a role in the pathogenesis of MCF. Virus-infected large granular lymphocytes (LGLs) were obtained from the tissues of rabbits infected with AlHV-1 or OvHV-2. These cells exhibited a similar proliferative response to IL-15 and to IL-2 in culture, but their content of the activated cytotoxic enzyme (BLT-esterase) was maintained at higher levels in the presence of IL-15 compared with IL-2. The LGLs did not express IL-15 mRNA or produce IL-15 protein. By contrast, there was abundant expression of IL-15 mRNA and protein in affected tissues. IL-15 production was associated with necrotic lesions of the mesenteric lymph node and appendix of OvHV-2-infected rabbits, but was not found in the same tissues of rabbits infected with AlHV-1 in which there were no necrotic lesions. The cellular source of the IL-15 was predominantly lymphoid cells that did not express B cell or monocyte-macrophage markers. Only a few IL-15+ cells (<10%) co-localized with pan-T cells or CD8+ T cells. The abundance of IL-15 in tissue with lesions of MCF suggests that this cytokine may have a role in the pathogenesis of MCF.

The A5 gene of alcelaphine herpesvirus 1 encodes a constitutively active G-protein-coupled receptor that is non-essential for the induction of malignant catarrhal fever in rabbits.

Many gammaherpesviruses encode G-protein-coupled receptors (GPCRs). Several in vivo studies have revealed that gammaherpesvirus GPCRs are important for viral replication and for virus-induced pathogenesis. The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) is carried asymptomatically by wildebeest, but causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species. The A5 ORF of the AlHV-1 genome encodes a putative GPCR. In the present study, we investigated whether A5 encodes a functional GPCR and addressed its role in viral replication and in the pathogenesis of MCF. In silico analysis supported the hypothesis that A5 could encode a functional GPCR as its expression product contained several hallmark features of GPCRs. Expression of A5 as tagged proteins in various cell lines revealed that A5 localizes in cell membranes, including the plasma membrane. Using [35S]GTPgammaS and reporter gene assays, we found that A5 is able to constitutively couple to alpha i-type G-proteins in transfected cells, and that this interaction is able to inhibit forskolin-triggered cAMP response element-binding protein (CREB) activation. Finally, using an AlHV-1 BAC clone, we produced a strain deleted for A5 and a revertant strain. Interestingly, the strain deleted for A5 replicated comparably to the wild-type parental strain and induced MCF in rabbits that was indistinguishable from that of the parental strain. The present study is the first to investigate the role of an individual gene of AlHV-1 in MCF pathogenesis.

Immunohistochemical study of experimental malignant catarrhal fever in rabbits.

Malignant catarrhal fever (MCF) is an often-fatal lymphoproliferative disease of a variety of ungulates that occurs worldwide. It is caused by either of the highly related but distinct gammaherpesviruses alcelaphine herpesvirus-1 (AlHV-1, wildebeest reservoir) or ovine herpesvirus-2 (OvHV-2, sheep reservoir). MCF in rabbits is an excellent model as it closely resembles the disease in susceptible ungulates that include cattle, deer and bison. In this study, newly available and previously characterized monoclonal antibodies specific for rabbit leucocyte differentiation molecules were used to perform a detailed immunohistochemical examination of both AlHV-1 MCF and OvHV-2 MCF in rabbits. Differences in the MCF caused by the two viruses included: less tissue necrosis and more lymphoid cell accumulations in AlHV-1 MCF compared with OvHV-2 MCF, and in particular marked tissue necrosis in the mesenteric lymph node, appendix and liver of OvHV-2-infected animals when compared with either other tissues in OvHV-2 MCF or AlHV-1 MCF lesions in any tissue. In both AlHV-1 MCF and OvHV-2 MCF, lymphoid cell accumulations in lymphoid and non-lymphoid tissues consisted mainly of T-cells with a corresponding absence of B-cells. CD8(+) T-cells accounted for a proportion of these in the non-lymphoid tissues, but there was evidence for the accumulation of an unidentified T-cell subset/subsets as well. This study extends our understanding of the mechanisms of immuno-pathogenesis of MCF.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test (k=0.86, n=95) results was found.

Cloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome.

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order Artiodactyla. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, mainly due to the fact that AlHV-1 becomes attenuated during passage in culture. In this study, these difficulties were overcome by cloning the entire AlHV-1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC). A modified loxP-flanked BAC cassette was inserted in one of the two large non-coding regions of the AlHV-1 genome. This insertion allowed the production of an AlHV-1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. The loxP-flanked BAC cassette was excised from the genome of reconstituted virions by growing them in permissive cells stably expressing Cre recombinase. Importantly, BAC-derived AlHV-1 virions replicated comparably to the virulent (low-passage) AlHV-1 parental strain and induced MCF in rabbits that was indistinguishable from that of the virulent parental strain. The availability of the AlHV-1 BAC is an important advance for the study of MCF that will allow the identification of viral genes involved in MCF pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

Genome re-arrangements associated with loss of pathogenicity of the gamma-herpesvirus alcelaphine herpesvirus-1.

The alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in ruminants. Previous work had shown that serial passage of AlHV-1 in culture resulted in genome alterations that are associated with a loss in pathogenicity. Here we have analysed the re-arrangements that occur in more detail. None of the observed re-arrangements was entirely consistent. However, they did all involve translocation of a similar region of DNA from around the centre of the genome to areas either next to or in between terminal repeat elements at either end of the genome. There was also a concomitant loss of the wild-type locus. These re-arrangements appeared to be associated with the loss of virulence and the appearance of cell-free virus.

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.

Subversion and piracy: DNA viruses and immune evasion.

During the co-evolution of viruses with their vertebrate hosts, the DNA viruses have acquired an impressive array of immunomodulatory genes to combat host immune responses and their hosts have developed a sophisticated immune system to contain virus infections. In order to replicate, the viruses have evolved mechanisms to inhibit key host anti-virus responses that include apoptosis, interferon production, chemokine production, inflammatory cytokine production, and the activity of cytotoxic T-cells, natural killer cells and antibody. In addition, some of the viruses encode cytokine or chemokine homologues that recruit or expand cell numbers for infection or that subvert the host cellular response from a protective response to a benign one. The specificity of the viral immunomodulatory molecules reflects the life cycle and the pathogenesis of the viruses. Herpesviruses achieve latency in host cells by inducing cell survival and protecting infected cells from immune recognition. This involves interference with cell signal transduction pathways. Many of the viral immunomodulatory proteins are homologues of host proteins that appear to have been pirated from the host and reassorted in the virus genomes. Some of these have unique functions and indicate novel or important aspects of both viral pathogenesis and host immunity to viruses. The specific example of orf virus infection of sheep is described.

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.

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.

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.

The cloning and expression of the cDNA for ovine stem cell factor (kit-ligand) and characterization of its in vitro haematopoietic activity.

The cDNA encoding the soluble form of ovine stem cell factor (SCF) has been cloned and expressed. The soluble protein is predicted to be 165/166 amino acids in length, one more than the human and murine SCFs with which it shares 87% and 81% identity respectively. Ovine SCF has 98.5%, 95% and 91% identity with cattle, pig and dog SCF, respectively. The recombinant ovine (rov) SCF protein has been expressed in Chinese hamster ovary (CHO) cells, purified, and its biological activity on ovine bone marrow cells compared with that of interleukin 3 (rovIL-3), granulocyte-macrophage colony-stimulating factor (rovGM-CSF), interleukin 5 (rovIL-5), human macrophage colony-stimulating factor (M-CSF) and human erythropoietin (epo). On its own rovSCF supported the development of small numbers of neutrophil, macrophage, eosinophil, granulocyte-macrophage, mixed cell phenotype, haemopoietic blast cell and basophilic granular cell colonies in a soft agar clonogenic assay. In combination with each of the above cytokines rovSCF supported an increase in the number and size of the lineage-specific colony types that were stimulated by the other cytokines on their own. In an assay for precursors of multipotential colony-forming cells (multi-CFC), rovSCF in combination with rovIL-3 (but neither cytokine alone) supported the development of these early haematopoietic progenitor cells.

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.

Poxvirus interference with the host cytokine response.

Poxviruses have evolved successfully to survive and replicate in a variety of species in the presence of an active host immune and inflammatory response. They manage this, at least in part, by the acquisition and modification of host immune and inflammatory response modulating genes. A proportion of these virulence genes encode homologues of host cytokines and cytokine receptors. These include soluble interferon, IL-1 beta, and TNF-alpha receptor homologues, that block the host cytokines. Other virulence gene products interfere with interferon signalling within cells and prevent the cleavage of biologically active IL-1 beta from its precursor protein. The parapoxvirus orf virus encodes a homologue of ovine IL-10 and a novel GM-CSF-binding protein. By studying poxvirus virulence proteins that interfere with host cytokine effector responses, important and novel aspects of the host immune and inflammatory response to infection have been revealed.

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-alpha) and type 2 (IFN-gamma) 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.

Eosinophil-specific biological activity of recombinant ovine interleukin-5.

Recombinant ovine interleukin-5 (rovIL-5) expressed from Chinese hamster ovary (CHO) cells was tested for cell-specific bioreactivity, in vitro, in a soft agar clonogenic assay and in an enzyme-based microassay for eosinophil potentiating activity (EPA). In soft agar assays, colony and cluster formation from sheep bone marrow cells (SBMC) incubated with rovIL-5 was significantly enhanced compared with SBMC incubated with control supernatants from mock-transfected CHO cells. Colony analysis at 14 days demonstrated that for three separate rovIL-5 preparations 45%, 61% and 66% of colonies were eosinophilic, as were 55%-71% of clusters. In contrast, no eosinophil colonies were detected in parallel control cultures. RovIL-5 was also shown to possess potent and dose-responsive EPA, on the basis of eosinophil peroxidase (EPO) and arylsulphatase (EAS) assay in 7 day SBMC cultures. This activity was inhibited in a dose-responsive manner by TRFK-5, a rat anti-murine IL-5 monoclonal antibody (MAb) previously shown to have cross-reactivity in the ovine EPA assay. The results demonstrate that rovIL-5 exhibited eosinophil-specific properties similar to those of IL-5 derived from other mammalian species.

The immune and inflammatory response to orf virus.

Orf virus is a zoonotic, epitheliotropic DNA parapox virus that principally infects sheep and goats. The fact that the virus can repeatedly reinfect sheep has provoked an interest in the underlying cellular, virological and molecular mechanisms for its apparent escape from the host protective immune response. The local immune and inflammatory response in skin and the cell phenotype and cytokine response in lymph analysed around a single lymph node are characteristic of an anti-viral response. An unusual feature is the dense accumulation of MHC Class II+ dendritic cells in the skin lesion. The function of these cells is not known. Orf virus virulence genes and activities have been identified that may interfere with the development of the host protective immune and inflammatory response.