Alcelaphine herpesvirus 1 glycoprotein B: recombinant expression and antibody recognition.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes fatal malignant catarrhal fever (MCF) in susceptible species including cattle, but infects its reservoir host, wildebeest, without causing disease. Pathology in cattle may be influenced by virus-host cell interactions mediated by the virus glycoproteins. Cloning and expression of a haemagglutinin-tagged version of the AlHV-1 glycoprotein B (gB) was used to demonstrate that the AlHV-1-specific monoclonal antibody 12B5 recognised gB and that gB was the main component of the gp115 complex of AlHV-1, a glycoprotein complex of five components identified on the surface of AlHV-1 by immunoprecipitation and radiolabelling. Analysis of AlHV-1 virus particles showed that the native form of gB was detected by mAb 12B5 as a band of about 70 kDa, whilst recombinant gB expressed by transfected HEK293T cells appeared to be subject to additional cleavage and incomplete post-translational processing. Antibody 12B5 recognised an epitope on the N-terminal furin-cleaved fragment of gB on AlHV-1 virus particles. It could be used to detect recombinant and virus-expressed gB on western blots and on the surface of infected cells by flow cytometry, whilst recombinant gB was detected on the surface of transfected cells by immunofluorescence. Recombinant gB has potential as an antigen for ELISA detection of MCF virus infection and as a candidate vaccine antigen.

The effect of the TLR9 ligand CpG-oligodeoxynucleotide on the protective immune response to alcelaphine herpesvirus-1-mediated malignant catarrhal fever in cattle.

We wished to determine the effect of of CpG ODN adjuvant on the magnitude and duration of protective immunity against alcelaphine herpesvirus-1 (AlHV-1) malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of cattle. Immunity was associated with a mucosal barrier of virus-neutralising antibody. The results showed that CpG ODN included either with emulsigen adjuvant and attenuated AlHV-1 (atAlHV-1) or alone with atAlHV-1 did not affect the overall protection from clinical disease or duration of immunity achieved using emulsigen and atAlHV-1. This is in contrast to other similar studies in cattle with BoHV-1 or cattle and pigs with various other immunogens. In addition to this, several other novel observations were made, not reported previously. Firstly, we were able to statistically verify that vaccine protection against MCF was associated with virus-neutralising antibodies (nAbs) in nasal secretions but was not associated with antibodies in blood plasma, nor with total virus-specific antibody (tAb) titres in either nasal secretions or blood plasma. Furthermore, CpG ODN alone as adjuvant did not support the generation of virus-neutralising antibodies. Secondly, there was a significant boost in tAb in animals with MCF comparing titres before and after challenge. This was not seen with protected animals. Finally, there was a strong IFN-γ response in animals with emulsigen and atAlHV-1 immunisation, as measured by IFN-γ secreting PBMC in culture (and a lack of IL-4) that was not affected by the inclusion of CpG ODN. This suggests that nAbs at the oro-nasal-pharyngeal region are important in protection against AlHV-1 MCF.

A novel spliced gene in alcelaphine herpesvirus 1 encodes a glycoprotein which is secreted in vitro.

Herpesviruses often contain cryptic, spliced genes that are not obvious from the initial in silico annotation. Alcelaphine herpesvirus 1 (AlHV-1) contains 72 annotated ORFs but there are also a number of gaps between these that may have protein-coding potential. Comparative analysis of coding potential between AlHV-1 and the related ovine herpesvirus 2 (OvHV-2) revealed a putative novel spliced gene that we have termed A9.5. Analysis of cDNA clones from AlHV-1-infected cells revealed three overlapping clones corresponding to A9.5 and the coding sequence was confirmed by reverse transcription PCR of RNA from AlHV-1-infected cattle tissues. The A9.5 gene was predicted to encode a secreted glycoprotein with molecular mass 19 kDa. Empirical analysis showed that a recombinant haemagglutinin-tagged A9.5 fusion protein was secreted from transfected cells and had a molecular mass of 45 kDa, which was reduced to 20 kDa by endoglycosidase F treatment, confirming that A9.5 was a secreted glycoprotein. In situ RNA hybridization showed that A9.5 was expressed in cells associated with malignant catarrhal fever (MCF) lesions in infected cattle. Detailed analysis of the available OvHV-2 sequences revealed an homologous gene (Ov9.5) with conserved splicing signals and predicted amino acid sequence features in both sequenced isolates of this related virus. We have therefore identified a novel spliced gene in two related macaviruses that is expressed in MCF lesions. Future work will determine its importance for the pathogenesis of disease.

Duration of protective immunity and antibody responses in cattle immunised against alcelaphine herpesvirus-1-induced malignant catarrhal fever.

Protection of cattle from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever (MCF) has been described previously, using an attenuated virus vaccine in an unlicensed adjuvant. The vaccine was hypothesised to induce a protective barrier of virus-neutralising antibody in the oro-nasal region, supported by the observation of high titre neutralising antibodies in nasal secretions of protected animals. Here we describe further analysis of this vaccine strategy, studying the effectiveness of the vaccine formulated with a licensed adjuvant; the duration of immunity induced; and the virus-specific antibody responses in plasma and nasal secretions. The results presented here show that the attenuated AlHV-1 vaccine in a licensed adjuvant protected cattle from fatal intranasal challenge with pathogenic AlHV-1 at three or six months. In addition, animals protected from MCF had significantly higher initial anti-viral antibody titres than animals that succumbed to disease; and these antibody titres remained relatively stable after challenge, while titres in vaccinated animals with MCF increased significantly prior to the onset of clinical disease. These data support the view that a mucosal barrier of neutralising antibody blocks infection of vaccinated animals and suggests that the magnitude of the initial response may correlate with long-term protection. Interestingly, the high titre virus-neutralising antibody responses seen in animals that succumbed to MCF after vaccination were not protective.

A host-independent role for Fasciola hepatica transforming growth factor-like molecule in parasite development.

The trematode parasite Fasciola hepatica causes chronic infection in hosts, enabled by an immunosuppressed environment. Both host and parasite factors are known to contribute to this suggesting that avoidance of immunopathology is beneficial to both parties. We have previously characterised a parasite transforming growth factor (TGF)-like molecule, FhTLM, that interacts with host macrophages to prevent antibody-dependent cell cytotoxicity (ADCC). FhTLM is one of many described helminth TGF homologues and multiple helminths are now known to utilise host immune responses as developmental cues. To test whether, or how, F. hepatica uses FhTLM to manipulate host immunity, we initially examined its effects on the CD4 T-cell phenotype. Despite inducing IL-10, there was no induction of FoxP3 within the CD4 T-cell compartment. In addition to inducing IL-10, a wide range of chemokines were elicited from both CD4 T-cells and macrophages. However, no growth or survival advantage was conferred on F. hepatica in our co-culture system when CD4 T-cells, macrophages, or eosinophils were tested. Finally, using RNA interference we were able to verify a host-independent role for FhTLM in parasite growth. Despite the similarities of FhTLM with other described helminth TGF homologues, here we demonstrate species-specific divergence.

Analysis of immune responses to attenuated alcelaphine herpesvirus 1 formulated with and without adjuvant.

The experimental vaccine for bovine malignant catarrhal fever consists of viable attenuated alcelaphine herpesvirus 1 (AlHV-1) derived by extensive culture passage, combined with an oil-in-water adjuvant, delivered intramuscularly. This immunisation strategy was over 80% effective in previous experimental and field trials and protection appeared to be associated with induction of virus-neutralising antibodies. Whether the vaccine virus is required to be viable at the point of immunisation and whether adjuvant is required to induce the appropriate immune responses remains unclear. To address these issues two studies were performed, firstly to analyse immune responses in the presence and absence of adjuvant and secondly, to investigate immune responses to vaccines containing adjuvant plus viable or inactivated AlHV-1. The first study showed that viable attenuated AlHV-1 in the absence of adjuvant induced virus-specific antibodies but the titres of virus-neutralising antibodies were significantly lower than those induced by vaccine containing viable virus and adjuvant, suggesting adjuvant was required for optimal responses. In contrast, the second study found that the vaccine containing inactivated (>99.9%) AlHV-1 induced similar levels of virus-neutralising antibody to the equivalent formulation containing viable AlHV-1. Together these studies suggest that the MCF vaccine acts as an antigen depot for induction of immune responses, requiring adjuvant and a suitable antigen source, which need not be viable virus. These observations may help in directing the development of alternative MCF vaccine formulations for distribution in the absence of an extensive cold chain.

Proteomic analysis of pathogenic and attenuated alcelaphine herpesvirus 1.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell.

Molecular cloning and characterization of Toll-like receptors 1-10 in sheep.

Toll-like receptors (TLRs) are pattern-recognition receptors that trigger innate immune responses and stimulate adaptive immunity. Currently, only partial information is available for sheep TLR genes. The aims of this study were to clone and sequence the coding regions of all 10 ovine TLR genes and compare the sequences with those of other mammalian species. The coding sequences for ovine TLRs 1-10 and the 3'-untranslated sequences for ovine TLR1, 6 and 10 have been obtained. Ovine TLR6 exhibited a distinctive 3'-end sequence that resembled a rare splice variant of bovine TLR6, but appeared to represent the major TLR6 transcript in the sheep. qRT-PCR confirmed the presence of TLR transcripts in blood mononuclear cells, alveolar macrophages, keratinocytes and lymph node tissues. Comparative sequence analysis showed that the sheep TLRs share high sequence similarity with the respective cattle, pig, human and mouse genes and are likely derived from the same ancestral sequence.

Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles.

Alcelaphine herpesvirus-1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF.

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.

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.

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.

The Effect of Intradermal Injection of GM-CSF and TNF-α on the Accumulation of Dendritic Cells in Ovine Skin.

Abstract- Intradermal recombinant ovine (rov) GM-CSF was associated with the accumulation over 5 DAys of MHC Class-II+ dendritic cells at the injection site, as well as increased numbers of neutrophils, eosinophils and lymphocytes when compared to control injection sites receiving heat-inactivated cytokines. Most of the dendritic cells were adjacent to the epidermis and dermal structures such as hair follicles, sweat glands and blood vessels. Fewer than 10% of the accumulating MHC Class-II+ dendritic cells expressed the CD1 antigen and none expressed detectable CD lib or CD lie antigens. They did not appear to be proliferating. Injection of rov TNF-α was associated with an increase in MHC Class-II+ dendritic cells that was significant only in the epidermis and only at 2 DAys following the first of four daily injections. Increased numbers of neutrophils and lymphocytes, but not eosinophils, were also recorded. Injection of rov TNF-α together with rov GM-CSF enhanced the accumulation of the MHC Class-II+, CD1+ subpopulation of dendritic cells compared to either cytokine alone. GM-CSF in particular is involved in an in vivo pathway supporting the accumulation of predominantly MHC Class-II+ CD1- dendritic cells which are a minor population in unchallenged skin. Résumé- Des injections intradermiques de GM-CSF recombinant ovin sont associées à l'accumulation en 5 jours de cellules dendritiques MHC classe II(+) au site de l'injection, ainsi qu'à une augmentation du nombre de neutrophiles, éosinophiles et lymphocytes, comparativement à ce qui est observe aux points d'injection témoins qui ont reçu des cytokines inactivées par la chaleur. La plupart des cellules dentritiques sont adjacentes à l'épiderme, aux follicules pileux, aux glandes sudoripares et aux vaisseaux sanguins. Moins de 10% des cellules dendritiques MHC classe II(+) accumulées expriment l'antigène CD1 et aucune n'exprime les antigènes CD11b et CD11c. Elles ne semblent pas proliférer. L'injection de TNF alpha recombinant ovin est associée à une augmentation des cellules dendritiques MHC classe II(+) qui est significative seulement dans l'épiderme, et seulement 2 jours suivant la première des quatre injections. Une augmentation du nombre de neutrophiles et de lymphocytes, mais pas des éosinophiles est également notée. L'injection simultanée de TNF alpha recombinant ovin et de GM-CSF recombinant ovin favorise l'accumulation d'une sous population de cellules dendritiques MHC classe II(+), CD1(+), comparativement à ce qui est observé en utilisant les cytokines séparément. GM-CSF en particulier est in vivo à l'origine de l'accumulation de cellules dendritiques MHC classe II(+), CD1(-) qui forment une population mineure dans la peau. [Haig, D. M., Hutchison, G., Green, I., Sargan, D., Reid, H. W. The effect of intradermal injection of GM-CSF and TNF-α on the accumulation of dendritic cells in ovine skin (Effect de l'injection intradermique de GM-CSF et de TNF alpha sur l'accumulation de cellules dentritiques dans la peau du mouton).

Analysis of the genetic diversity of ovine herpesvirus 2 in samples from livestock with malignant catarrhal fever.

In order to define better virus isolates from animals with malignant catarrhal fever (MCF), segments of three genes of ovine herpesvirus-2 were amplified from diagnostic samples representing MCF cases with a range of clinical presentations in cattle, including head and eye, alimentary and neurological. The variation within each gene segment was estimated by DNA sequencing, which confirmed that the newly-annotated Ov9.5 gene was significantly more polymorphic than either of the other loci tested (segments of ORF50 and ORF75), with alleles that differed at over 60% of nucleotide positions. Despite this, the nine Ov9.5 alleles characterised had identical predicted splicing patterns and could be translated into Ov9.5 polypeptides with at least 49% amino acid identity. This multi-locus approach has potential for use in epidemiological studies and in charactering chains of infection. However there was no association between specific variants of OvHV-2 and the clinical/pathological presentation of MCF in the cattle analysed.

Identification of immuno-reactive capsid proteins of malignant catarrhal fever viruses.

Malignant catarrhal fever (MCF) is a fatal disease of cattle and other ungulates caused by certain gamma-herpesviruses including alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). An attenuated virus vaccine based on AlHV-1 has been shown to induce virus-neutralising antibodies in plasma and nasal secretions of protected cattle but the targets of virus-specific antibodies are unknown. Proteomic analysis and western blotting of virus extracts allowed the identification of eight candidate AlHV-1 virion antigens. Recombinant expression of selected candidates and their OvHV-2 orthologues confirmed that two polypeptides, the products of the ORF17.5 and ORF65 genes, were antigens recognised by antibodies from natural MCF cases or from AlHV-1 vaccinated cattle. These proteins have potential as diagnostic and/or vaccine antigens.

Host gene expression changes in cattle infected with Alcelaphine herpesvirus 1.

Malignant catarrhal fever is a lymphoproliferative disease of cattle and other ungulates caused by infection with gamma-herpesviruses of the genus Macavirus. These viruses do not establish a productive infection but instead replicate in a cell-associated fashion in T lymphocytes, leading to systemic immune dysregulation and a generally fatal outcome. Despite significant progress in understanding the pathology of this disease, its pathogenesis remains unclear. To identify genes and pathways affected in clinical MCF, sixteen bovine GeneCHIP microarrays were used to assay RNA from kidney and lymph node of four MCF-affected and four control Bos taurus steers. This is the first expression study of AlHV-1-MCF in the bovine host. Over 250 genes showed significant changes in gene expression in either lymph node or kidney, while expression of 35 genes was altered in both tissues. Pathway and annotation analysis of the microarray data showed that immune response and inflammatory genes were up-regulated in the kidney while proliferation-associated transcripts were additionally increased in the lymph node. The genes that showed the largest expression rises in both diseased tissues included cytotoxic enzymes and pro-inflammatory chemokines. These data are consistent with disease-induced stimulation of inflammatory responses involving interferon-γ, including cytotoxic T cell recruitment and activation in peripheral tissues containing virus-infected cells. However it remains unclear whether the tissue damage in MCF lesions is due entirely to the activity of infected cells or whether uninfected T cells, recruited and activated at lesion sites through the action of infected cells, contribute to the pathogenesis of MCF.

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.

Ovine herpesvirus 2 structural proteins in epithelial cells and M-cells of the appendix in rabbits with malignant catarrhal fever.

Sheep-associated malignant catarrhal fever (MCF), caused by Ovine herpesvirus 2 (OvHV-2), is a usually fatal disease of various ruminants and swine. A system for propagation of OvHV-2 in vitro has not yet been identified, although persistently infected cells have been derived from diseased animals and used to establish an animal model in rabbits. OvHV-2 structural proteins have not been detected in diseased animals and the pathogenesis of OvHV-2 infection is poorly understood. Recently, the genomic sequence of OvHV-2 has been determined, which allowed to predict the amino acid sequences of putative OvHV-2 structural proteins. Based on those predictions, we have generated antisera against two putative structural proteins (ORF43 and ORF63) of OvHV-2 in order to detect sites of active virus replication in experimentally OvHV-2-infected rabbits with signs of MCF. Although histological lesions typical of MCF were detected in multiple tissues, those sera detected viral capsid and tegument antigens exclusively in the appendix but not in other tissues of rabbits with MCF. More specifically, those viral proteins were detected in epithelial cells as well as in M-cells. However, in situ hybridization revealed that ORF63 mRNA was present in epithelial cells of infected rabbits but not in M-cells. Our data suggest that active OvHV-2 replication takes place in certain tissues of animals with MCF and that M-cells may play a role in the pathogenesis of MCF.

An immunisation strategy for the protection of cattle against alcelaphine herpesvirus-1-induced malignant catarrhal fever.

The aim of this study was to stimulate immunity in the oro-nasal-pharyngeal region of cattle to protect them from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever. Attenuated C500 strain AlHV-1 was used along with Freund's adjuvant intramuscularly (IM) in the upper neck region to immunise cattle. Virulent C500 strain AlHV-1 was used for intranasal challenge. Nine of ten cattle were protected. Protection was associated with high levels of neutralising antibody in nasal secretions. Some protected animals showed transient low levels of viral DNA in blood samples and in one lymph node sample after challenge whereas viral DNA was detected in the blood and in lymph node samples of all animals with MCF. This is the most promising immunisation strategy to date for the control of malignant catarrhal fever.

A captured viral interleukin 10 gene with cellular exon structure.

We have characterized a novel, captured and fully functional viral interleukin (IL)-10 homologue ((OvHV)IL-10) from the gammaherpesvirus ovine herpesvirus 2. Unlike IL-10 homologues from other gammaherpesviruses, the (OvHV)IL-10 peptide sequence was highly divergent from that of the host species. The (OvHV)IL-10 gene is unique amongst virus captured genes in that it has precisely retained the original cellular exon structure, having five exons of similar sizes to the cellular counterparts. However, the sizes of the introns are dramatically reduced. The (OvHV)IL-10 protein was shown to be a non-glycosylated, secreted protein of M(r) 21 000 with a signal peptidase cleavage site between amino acids 26 and 27 of the nascent peptide. Functional assays showed that (OvHV)IL-10, in a similar way to ovine IL-10, stimulated mast cell proliferation and inhibited macrophage inflammatory chemokine production. This is the first example of a captured herpesvirus gene retaining the full cellular gene structure.