Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004.

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our understanding of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field.

The equid herpesvirus-1 gene 63 is expressed as a leaky late (gamma 1) transcript and is nonessential for replication in vitro.

The regulation of Equid herpesvirus-1 (EHV-1) gene 63 was investigated using molecular expression studies and its role in viral growth was identified by constructing a gene 63 mutant virus. Metabolic inhibitors were used to show that EHV-1 gene 63 is expressed as a leaky-late (gamma 1) transcript. Transient transfections and subsequent chloramphenicol acetyltransferase (CAT) reporter assays showed that gene 63 was transactivated by EHV-1 gene 64 (immediate early) protein. An EHV-1 gene 63 mutant virus, where the LacZ gene was inserted into the mutated gene 63 open reading frame, showed that gene 63 protein was not essential for efficient EHV-1 growth in tissue culture. These findings indicate that the animal alpha herpesviruses may have evolved different pathways leading to replication.

Time of transforming growth factor beta 1 inoculation alters the incubation of BSE in mice.

Groups of 20 C57BL/6J mice (10 males and 10 females) were given BSE strain 301C i.p. and subsequently given 2 microg recombinant human TGFbeta1 s.c. at single or multiple times. There was a significant positive correlation between the day of TGFbeta1 administration and incubation time; the later TGFbeta1 was administered after BSE inoculation the longer the incubation time became. The administration of TGFbeta1 at any time point did not significantly alter the distribution or severity of pathology. The effects of TGFbeta1 on BSE pathogenesis appears to be dependent upon its time of administration; early administration shortens the incubation time and late administration lengthens the incubation time.

Clusterin shortens the incubation and alters the histopathology of bovine spongiform encephalopathy in mice.

Clusterin accumulates in significant quantity in prion protein lesions associated with bovine spongiform encephalopathy (BSE) and we therefore sought to elucidate its ability to alter BSE pathogenesis and incubation time by comparison of wild type C57BL/6J mice and clusterin knock out (ko) mice. The ko mice had a 40 day increase in mean incubation time compared to wild type mice. PrP deposition in the medulla was less aggregated in clusterin knock out mice when compared to wild type BSE infected mice and a more marked astrocytosis, as determined by GFAP staining, was evident. The vacuolation profiles did not differ between the two strains of mice. Taken together these results suggest that clusterin alters the extracellular deposition of PrP(BSE) and accelerates BSE pathogenesis.

Impaired motor coordination on static rods in BSE-infected mice.

Scrapie and bovine spongiform encephalopathy (BSE) are both progressive neurodegenerative diseases that are transmissible to mice. The onset of clinical symptoms is more subtle and variable in murine BSE than in murine scrapie. Assessment of behavioural changes that occur throughout disease would aid early diagnosis of disease so that more consistent end points could be made and potential therapies could be investigated. C57BL/6J mice inoculated via the intraperitoneal route with 301C BSE or control inoculum were monitored on a fortnightly basis. The end point was when a mouse showed clinical signs as opposed to behavioural signs of BSE for two consecutive observations. Significant loss of motor function, as assessed by mice balancing on a static rod, was observed consistently from approximately 40 days prior to death. No significant differences in home cage activity (locomotion, rearing) or cognitive function (T-maze alternation) were observed. However, there was an increase in digging by BSE-infected mice from an early stage. This data will aid the standardisation of behavioural tests to characterise and assess the onset of BSE.

The pathogenicity in mice of respiratory, abortion and paresis isolates of equine herpesvirus-1.

Eleven isolates of equine herpesvirus-1 (subtype 1) all infected the brain following intracerebral inoculation of 2 d.o. mice. Most isolates were from cases of paresis, abortion or respiratory disease in the U.K., but established strains were also included. They divided into two subgroups. The 5 less pathogenic isolates were characterized by being restricted predominantly to the olfactory lobes. The 6 pathogenic isolates included the three known to cause equine paresis and were detected in neurones throughout the brain as well as giving rise to viraemia and infecting bronchial and renal epithelium and lymphoid cells in the spleen. Five respiratory isolates of subtype-2 were not recovered from inoculated mice.

The effect of antibody and complement on the expression of herpesvirus of bovine malignant catarrhal fever in cultured rabbit lymphocytes.

Using indirect immunofluorescence with a hyperimmune calf serum, a virus-induced antigen was demonstrated on the surface of lymphocytes expression intracellular malignant catarrhal fever virus antigens. Antibody to the antigen was also detected in terminal sera of both cattle and rabbits. Antisera did not restrict virus expression in explanted lymph nodes unless they were supplemented with two to four units of lytic complement per ml culture. While human, bovine and guinea pig complements caused immune lysis of infected lymphocytes, rabbit complement was ineffective. The relevance of the findings in the pathogenesis of the lymphoid proliferation caused by MCFV is discussed.

Immune complexes associated with infection of cattle by the herpesvirus of malignant catarrhal fever.

Associated with the fatal lymphoid proliferation induced by the herpesvirus of African malignant catarrhal fever (MCFV) was the deposition of immune complexes comprising immunoglobulin, complement and conglutinin in the kidneys of cattle with terminal pyrexia due to infection with this virus. Such deposits were common in glomeruli and were also seen in renal arteries with an without lesions. Absence of lytic complement and a significant depletion of conglutinin in terminal sera confirmed the activation of complement. However, virus antigen was only rarely detected and virus-specific antibody could not be demonstrated in the deposits. These disparities and the importance of the observations are discussed.

Porcine cytomegalovirus (PCMV) in early gestation.

Following intranasal exposure to PCMV at or within 48 h of coitus transplacental infection occurred in two groups of gilts. Five out of 22 embryos were infected in the first group but only 2 out of 63 in the second. A more rapid immune response as measured by circulating antibody was probably instrumental in abrogating infection in the second group. In the infected embryos the virus localized in leptomeningeal cells, hepatic sinusoidal cells, peritoneal macrophages, periosteal cells and occasional alveolar cells, but the placenta did not appear to be a primary site of viral replication.

Immunoprecipitation of viral polypeptides of equid herpesvirus 1 and 4 by serum from experimentally infected ponies.

Sera from two sibling groups of ponies experimentally infected with Equid herpesvirus 1 or 4 (EHV-1 or 4) were used to investigate which viral polypeptides (VPs) of EHV-1 and EHV-4 were recognised. Recognition was detected as early as 8 d.p.i. and thereafter. The polypeptides of EHV-1 (labelled with 35S-methionine) immunoprecipitated (IIP) by sera from both groups had Mr of 148, 138, 123, 117, 110, 77-79, 70, 55, 49-50, 47, 40 and 35-37 kDa respectively. Of these VP148K (VP9 nucleocapsid) gave the maximum precipitation, followed by 117 and 77-79 kDa. The latter were confirmed by monoclonal antibodies as the gB homologue of Herpes-simplex virus (HSV). With EHV-4 the homologous VPs precipitated were similar to those of EHV-1. However, instead of the precipitated VP55K of EHV-1, there were two faint bands of Mr 60 and 55 kDa, neither of significant density. Bands at 123 and 70 kDa were absent. High MW polypeptides (>200 kDa) were not significant and infrequently seen with both viruses. Labelling EHV-1 with 3H glucosamine indicated that viral glycoproteins (VGPs) at an Mr of 79-88 kDa (equivalent to gB and gC) were most commonly recognised in homologous EHV-1 IIP and at 83 kDa (gC) in heterologous IIP. The EHV-4 immunoprecipitated VGPs were at 230-300 kDa with bands at 290 kDa and 250 kDa. Also detected were bands at 100, 123, 79-88, 58-61K and 54-55 kDa. The 79-88 kDa polypeptides gave maximum density and were considered as homologues of HSV gB and gC. Thus the overall profile indicated that following experimental infection the major nucleocapsid protein of 148K, and the gB analogues of 117 and 77 kDa were the most antigenic in experimental infections of ponies with either EHV-1 or 4 and that these showed reciprocal precipitation.

The effect of EHV-1 infection upon circulating leucocyte populations in the natural equine host.

It has been suggested that EHV-1 infection may perturb immune responsiveness in the natural equine host. The mechanism underlying this phenomenon is not clear, but disturbances of circulating leucocyte populations could contribute. In order to objectively assess the nature of the haematological changes provoked by EHV-1 infection, two groups of conventionally-maintained Welsh mountain ponies were challenge-infected intra-nasally with the Ab4 isolate of EHV-1. These groups were controlled by similarly-sized groups of non-infected ponies. All data generated was subjected to rigorous statistical analysis. Whole leucocyte count, neutrophil count, lymphocyte count, pan T cell count (RVC1 + cells-putative CD5 homologue), T cell subset count (RVC3 + cell-putative CD8 homologue), RVC2 + cells (putatively class II MHC+) and B cell count were recorded in experimental and control subjects at frequent intervals post-infection via flow cytometry. The principal abnormalities post-infection were T cell lymphopaenia, neutropaenia and the appearance of blastic cells of undetermined lineage. This study underlined the variability of EHV-1 infection in the natural, outbred equine host.

Superinfection with porcine cytomegalovirus initiating transplacental infection.

Four sows with circulating antibody were exposed to porcine cytomegalovirus. Virus was detected in 8 of 24 foetuses by immunofluorescence and/or virus isolation from 2 sows with low levels of antibody. In 6 of the infected foetuses, the virus was in capillary endothelium and macrophages of the lung, and was associated with interlobular oedema in 2 of these. Virus was also detected in the nasal mucosa, spleen and brain. The majority of the virus-positive foetuses were grossly normal.

Cross-hybridization of equid herpesvirus-2 (EHV-2) and herpes simplex virus-1 (HSV-1) genes to equid herpesvirus-1 (EHV-1).

In contrast to previous findings, the Ab4 isolate of equid herpesvirus-1 (EHV-1) was shown to share homology with the G9 isolate of equid herpesvirus-2 (EHV-2). Using Southern blotting and stringent hybridization conditions, a significant proportion of this cross-hybridization was identified by the immediate-early gene-3 (IE-3) probe from herpes simplex virus-1 (HSV-1). The HSV-1 UL48 gene probe (encoding the IE gene transactivating protein VmW65, which is also known as alpha-TIF or VP16) was used to identify and isolate its counterpart in EHV-1. The relevance of shared homology to transactivation is being investigated.

Equid herpesvirus 1: platelets and alveolar macrophages are potential sources of activated TGF-B1 in the horse.

Cell mediated responses to Equid herpesvirus 1 (EHV-1) are of short duration in vivo and require considerable expansion to be detected in vitro. Raised serum levels of active transforming growth factor B (TGF-B1) have been shown to depress proliferative T cell responses in experimental infections with EHV-1 in ponies. The present work indicates that latent transforming growth factor B (TGF-B1) is present in circulating platelets, lymph node, bronchial epithelium and alveolar macrophages. Activation of platelets in vitro by thrombin resulted in the release of latent TGF-B1 from platelets, with a pg level of conversion to active TGF-B1, but virus alone did not activate TGF-B1. Exposure of circulating leucocytes to EHV-1 in vivo or in vitro does not result in detection of active TGF-B1 above residual levels that could be attributed to activation of platelets by manipulation. However, alveolar macrophages obtained by lavage at autopsy yield both latent and active TGF-B1 in ng quantities. Bronchial epithelium, and mesenteric lymph node leucocytes had equivalent levels of latent TGF-B1, but horses varied as to whether these tissues were a source of activated TGF-B1 and as to whether EHV-1 activated TGF-B1.

Equine herpesvirus type 1 infects dendritic cells in vitro: stimulation of T lymphocyte proliferation and cytotoxicity by infected dendritic cells.

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses. As with other herpesviruses, cell-mediated immunity is considered important for both recovery and protection. Although virus-specific T-cell proliferation and cytotoxicity can be detected following in vivo infection, little is known about the role of antigen presenting cells such as dendritic cells (DCs) in these processes. Peripheral blood DCs were shown to express the viral glycoprotein gB perinuclearly following exposure to EHV-1 in vitro, demonstrating EHV-1 replication within them. Co-culture of infected DCs or their supernatants with a susceptible cell line (RK13) demonstrated that EHV-1 infection was productive. In vitro-infected DCs showed cytopathic effects, including loss of viability and syncytial formation. However, they were superior to other antigen presenting cells in stimulating both peripheral blood T-cell proliferation and cytotoxicity. Although ponies which had been intranasally infected with EHV-1 exhibited T-cell proliferation to live virus presented on DCs, the responses began to decline as early as 15 weeks and cease at 22 weeks post-in vivo infection. Cytotoxic responses were not detected 35 weeks after the first intranasal infection but were seen again 7 weeks following a second infection. These findings show that equine DCs, which are infected with EHV-1 in vitro, can stimulate memory T-cell responses but appear unable to circumvent the short-lived memory response found following this infection in vivo.

Isolation and characterisation of equine dendritic cells.

Despite their important role in initiating T-cell responses in other species, dendritic cells have not been studied in the horse. A method for isolating blood dendritic cells by adherence and metrizamide gradients was adapted to equine cells. A number of monoclonal antibodies (mAbs), including some which label dendritic cells in other species, were tested for immunochemical reactivity with the isolated blood dendritic cells, and sections of lymph node and spleen. 62 +/- 6% of the isolated blood cells were MHC Class II positive and had typical dendritic cell morphology and only 4 +/- 2% contained non-specific esterase, a marker of mature macrophages. These dendritic cells also expressed MHC Class I, LFA-1, EqWC1 and EqWC2. Amongst the potentially cross-reactive antibodies a mAb against bovine CD1b was the most interesting by staining lymph node, but not blood, dendritic cells. Monoclonal antibodies against equine CD5 (T-cells), surface immunoglobulin (B-cells) and macrophages (CZ2.2) were used to enumerate the contaminating cells in preparations from blood by flow cytometry. 39 +/- 7% of the cells did not express T and B cell markers or CZ2.2 but were large and MHC Class II positive. Comparison of immuno-chemistry and flow data, together with examination of alveolar macrophages and adhered blood cells, all support the view that CZ2.2 detects a myeloid marker not seen on mature macrophages and possibly shared with dendritic cell precursors. The functional capacity of the isolates was assessed in terms of their stimulating ability in the mixed leukocyte reaction (MLR). Dendritic cell enriched isolates were more potent stimulators of MLRs than peripheral blood mononuclear cells or adherent cells. Thus equine dendritic cells isolated from blood express high levels of MHC Class I and II and LFA-1 and stimulate a vigorous MLR. They do not express markers characterising T and B cells but, by virtue of expression of the equine macrophage marker CZ2.2, appear closely related to mononuclear phagocytes.

Report of the First International Workshop on Equine Leucocyte Antigens, Cambridge, UK, July 1991.

The First International Workshop on Equine Leucocyte Antigens was organized and convened for the purposes of identifying immunologically relevant cell surface molecules of equine leucocytes and establishing a system of nomenclature for those molecules. Participating members of the workshop represented the majority of laboratories world-wide engaged in the tasks of production and characterization of equine leucocyte and lymphocyte markers using monoclonal antibodies. The workshop confirmed the identification of several equine CD molecules described previously by individual laboratories, and in addition recognized antibodies identifying new CD molecules. The workshop also succeeded in fostering co-operation between laboratories around the world which study equine immunobiology. Equine CD molecules identified by the current battery of monoclonal antibodies include EqCD2, EqCD4, EqCD5, EqCD8, EqCD11a/18, EqCD13 and EqCD44. Other antibodies are markers for MHC class I and class II molecules, for B cells, granulocytes, macrophages, T cell subsets distinct from those defined by CD4 and CD8, and other sub-populations of horse leucocytes that do not have obvious counterparts in humans, rodents, or other species. Despite the progress made in the first workshop, there are still substantial gaps in the armory of reagents available to study equine leucocyte biology, and further definition of the structure, function, and genetics of the antigens identified by the workshop clusters (WC1, WC2 etc.) and other molecules of immunological importance will be a goal of future workshops. The study of equine immunobiology and resistance to disease also urgently requires the development of tools to study equine immunoglobulins and cytokines, and these needs will provide ample scope for future studies.

The role of endothelial cell infection in the endometrium, placenta and foetus of equid herpesvirus 1 (EHV-1) abortions.

One of three mares in the last trimester of pregnancy became paraplegic 7 days after experimental infection with EHV-1 and was killed 10 days after infection (d.p.i.). The other two mares aborted foetuses at 12 and 14 d.p.i. In the first mare, virus was detected by immunofluorescence (IIF) and immunoperoxidase (IP) staining in endothelial cells of the endometrium, placenta and umbilical vein, but not in any other foetal tissues. In the experimentally aborted foetuses, and in two other independent field cases of abortions, endothelial cell infection was also detected in the foetuses, both in major blood vessels and in capillaries or sinusoidal cells associated with parenchymal lesions. In these four cases there were also positive endothelial placental lesions detected by IIF or IP, although it was not always possible to isolate virus from these tissues, as it was from the foetuses. The evidence suggests that infection of maternal endothelial cells has a major role in the pathogenesis of abortion, and that endothelial cells are also involved in dissemination of virus within the foetus.

Neuritis of the cauda equina in the horse.

Ultrastructural lesions of the cranial nerves and their ganglia and the autonomic nervous system from 5 cases of neuritis of the cauda equina in the horse are described. They include lysosomal inclusions within the semilunar, geniculate and sympathetic chain ganglia, granulomatous involvement of the coeliaco-mesenteric ganglion and accumulation of axonal organelles in unmyelinated fibres of the great splanchnic nerve, sympathetic chain and oesophageal vagus.

Clusterin in bovine spongiform encephalopathy (BSE).

Clusterin mRNA, detected in increased quantities in the cervical spinal cord of cattle with bovine spongiform encephalopathy (BSE), was localized mainly in the neuroglia (including astrocytes) of the lateral and ventral areas of white matter. Axonal degeneration was also observed in these areas. The dorsal horns of the spinal cord in which BSE prion protein (PrP(BSE)) was deposited did not exhibit strong clusterin "up-regulation" but showed increased clusterin immunolabelling with a punctate distribution in the neuropil. Labelling of adjacent sections of the grey matter in BSE-affected spinal cord and thalamus demonstrated that the clusterin was deposited in association with extracellular PrP(BSE).