Pulmonary angiocentric lymphoma (lymphomatoid granulomatosis) in a donkey.

A 36-year-old donkey developed dyspnoea, pyrexia, hypoalbuminaemia and oedema. Following continued clinical deterioration the donkey was humanely destroyed. Grossly, there were numerous nodules (5-10mm) scattered throughout the lung. Microscopically, the lung was infiltrated by an angiocentric and bronchocentric to diffuse mixed population of small mature and atypical lymphocytes, histiocytes, plasma cells and fewer eosinophils. The infiltrate was composed of numerous small mature and fewer atypical CD3(+) T lymphocytes. Low numbers of CD20(+) and CD79a(+) B cells, some atypical, accompanied the T cells. These infiltrates were consistent with an angiocentric lymphoma and resembled lymphomatoid granulomatosis, an Epstein-Barr virus (EBV)-associated human tumour. Immunohistochemistry for EBV latent membrane protein and polymerase chain reaction analysis for equine gamma herpesvirus DNA were negative. To the authors' knowledge this is the first case of angiocentric lymphoma reported in a donkey and the first case of lymphomatoid granulomatosis-type disease in an animal in which possible concurrent infection with a gamma herpesvirus has been investigated.

The alcelaphine herpesvirus-1 ORF 57 encodes a nuclear shuttling protein.

Alcelaphine herpesvirus-1 (AlHV-1) is a gamma(2) rhadinovirus associated with Malignant Catarrhal Fever (MCF) in cattle. ORF 57 is well conserved among gammaherpesviruses and it has been shown that the ORF 57 gene products of Herpesvirus Saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) play an important role in regulating viral gene expression. The AlHV-1 ORF 57 gene product has not been characterized. In the accompanying paper we have demonstrated that AlHV-1 ORF 57 encodes an immediate early protein that acts as a regulator of gene expression. The ORF 57 gene product has an up-regulatory effect only on another immediate early gene product encoded by ORF 50. Here we show that the ORF 57 gene product is a nuclear protein. When ORF 57 was fused to the gene encoding Enhanced Green Fluorescent Protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the nucleolar phosphoprotein C23. The nuclear localisation signal of ORF 57 gene product was located at the N-terminus. The ORF 57 gene product travels from nucleus to the cytoplasm, where it accumulates during Actinomycin D treatment. The domain involved in nuclear shuttling was also localised at the N-terminal region of the protein. Thus in common with homologues in other herpesviruses the AlHV-1 ORF 57 gene product is a nuclear cytoplasmic shuttling protein which may play a role in export of viral mRNAs from the nucleus of infected cells.

Alcelaphine herpesvirus-1 open reading frame 57 encodes an immediate-early protein with regulatory function.

Alcelaphine herpesvirus-1 (AlHV-1) is the causative agent of Malignant Catarrhal fever, a lymphoproliferative and degenerative disease of large ruminants and ungulate species. The Alcelaphine Herpesvirus-1 gene product encoded by open reading frame 57 (ORF 57) is the positional homologue of the ORF 57 of Herpes Virus Saimiri (HVS), Kaposi's Sarcoma associated herpesvirus (KSHV) and Murine Gammaherpesvirus 68 (MHV 68), the Epstein-Barr virus BMLF1 gene, the herpes simplex virus (HSV-1) ICP 27 and the IE 4 gene of Varicella Zoster virus (VZV). In these viruses the ORF 57 gene product is expressed very early and encodes a regulatory protein, which is essential for viral replication acting both at the transcriptional and post-transcriptional levels. The function of ORF 57 gene product in the life cycle of AlHV-1 however remains unknown. Here we examined the expression of this gene and the function of its product. We have demonstrated that it is expressed very early in infection and have shown that the ORF57 gene product activates the promoter of another classical transactivator gene ORF50. It activates ORF50 promoter driving expression of an intron-less reporter gene to 50 fold and does not have any effect on an intron-containing reporter gene driven by the ORF 50 promoter. The 50 fold increase in the luciferase activity was not correlated with a similar fold increase in the luciferase RNA levels indicating that ORF 57 protein acts at a post-transcriptional level to regulate gene expression.

The effects of gene gun delivered pIL-3 adjuvant on skin pathology and cytokine expression.

The aim of this study was to investigate skin immunopathology following gene gun delivery of plasmid-encoding interleukin 3 (pIL-3) and hence explore the possible mechanisms of its adjuvant activity. Using the sheep as the experimental model, expressible pIL-3 was administered to the epidermis and the dermal/epidermal junction and its effects on the skin were assessed by histopathology, immunohistology and quantitative RT-PCR for a range of pro-inflammatory and immune response polarizing cytokines. Delivery of both functional and non-functional plasmids caused an acute inflammatory response with the infiltration of neutrophils and micro-abscess formation; however, the response to pIL-3 was more severe and was also associated with an early (24 h) infiltration of B cells and a later accumulation of CD172a-/CD45RA+ dendritic cells (DC). In terms of cytokine transcript expression, an early TNFalpha response was stimulated by gene gun delivery of plasmid-associated gold beads, which coincided with an immediate infiltration of neutrophils. However, only pIL-3 triggered the short-lived expression of IL-3 (peaking at 6 h) and significant long-term increases in both TNFalpha and IL-1beta. pIL-3 did not affect the expression of the immune response polarizing cytokines, IL-10 and IL-12.

Gene gun-delivered pGM-CSF adjuvant induces enhanced emigration of two dendritic cell subsets from the skin.

Two subsets of sheep afferent lymph dendritic cells (DC) are defined by the differential expression of CD172a and CD45RA. The majority (~70%) of CD172a(+) subset is CD45RA/CD11c(+)/CD207(+)/TLR4(+). The CD172a(-) DC are CD45RA(+)/CD207(-) and express low levels of CD11c and CD86. Real-time RT-PCR showed that CD172(+) DC produce IL-1beta and IL-10 and high levels of IL-18 but almost no IL-12p40; CD172a(-) DC express IL-12p40 but no IL-10 and low levels of IL-1beta and IL-18. Gene gun-delivered granulocyte-macrophage colony-stimulating factor (pGM-CSF) caused an early rise in the output of CD172a(+) DC, changes to DC phenotype and significant increases in the levels of expression cytokine transcripts. However, pGM-CSF did not affect any qualitative changes to cytokine expression, CD172a(+) DC remained IL-10(+)/IL-12p40(-) and the CD172(-) DC remained IL-10(-)/IL-12p40(+).

The effect of gene gun-delivered pGM-CSF on the immunopathology of the vaccinated skin.

The aim of this study was to investigate the skin immunopathology of gene gun-delivered plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) and hence explore the possible mechanisms of its adjuvant activity. Using sheep as the experimental model, expressible pGM-CSF was administered to the epidermis and the dermal/epidermal junction and its effects on the skin were assessed by histopathology, immunohistology and quantitative RT-PCR for a range of pro-inflammatory and immune response-polarizing cytokines. Both functional and non-functional plasmids caused an acute inflammatory response with the infiltration of neutrophils and micro-abscess formation; however, the response to pGM-CSF was more severe and was also associated with the accumulation of eosinophils, immature (CD1b(-)/CD172a(-)) dendritic cells and B cells. In terms of cytokine expression, an early TNF-alpha response was stimulated by gene gun delivery of plasmid-associated gold beads, which coincided with an immediate infiltration of neutrophils. However, only pGM-CSF triggered the short-lived expression of GM-CSF (peaking at 4 h) and significant long-term increases in both TNF-alpha and IL-1beta. pGM-CSF did not affect the expression of the immune response-polarizing cytokines, IL-10 and IL-12.

Herpes virus latency in sensory ganglia--a comparison with endogenous neuronal gene expression.

Central to infection by a majority of DNA viruses is the expression of encoded proteins that modify cell cycle. Viruses such as SV40 and Adenovirus viruses encode proteins that interact directly, or indirectly, with key cell cycle proteins such as CBP300 and the retinoblastoma gene product. However, neurons do not have a cell cycle as we generally describe it and this is also reflected in the difficulty in obtaining immortalised neuronal cultures. The replication strategies of viruses that infect post-mitotic cells such as neurons may be different from infection of other somatic cells. The life cycle for viral latency or slow infection of neurons appears to involve silencing or restricting expression of the viral genome until such times as dictated by the environment. These signals from the environment usually reflect cell stress, otherwise the cell appears to tolerate the existence of the virus genome. We will review the genomic structure of alphaherpesviruses in neurons and transcriptional control mechanisms that may regulate expression. Where appropriate we will contrast and compare virus and endogenous neuronal gene expression.

Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector.

The ability to manipulate the expression of genes within neurons provides unique opportunities to study the role of individual gene products in nervous system function. Virus vectors are a potentially rapid tool for the experimental manipulation of gene expression in the mammalian nervous system. However, a block to the use of virus vector systems in neurobiology is often the lack of cell-specific expression of the gene within the nervous system, and the immune and inflammatory responses to both the virus vector and the delivered gene. We have generated an adeno-associated virus vector that exploits the restricted expression pattern of the rat preprotachykinin-A promoter to support reporter gene expression. We demonstrate that this virus has a neuronal-specific expression pattern. Moreover, it is shown for the first time that the proximal rat preprotachykinin-A promoter is nerve growth factor inducible. This virus will be a useful tool to (i) modify neuronal phenotype by expressing therapeutic molecules or antisense nucleic acid and (ii) dissect the signal transduction pathways that regulate promoter function in vivo.

Behavioural changes in the rat following infection with varicella-zoster virus.

Following the establishment of a chronic varicella-zoster virus infection in the rat, behavioural allodynia and hyperalgesia were observed in the injected, but not the contralateral hind limb up to 33 days post-infection. This model may prove useful in investigating mechanisms involved in the establishment of post-herpetic neuralgia.

Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.

The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses.

Identification of a novel multifunctional structural domain in the herpes simplex virus type 1 genome: implications for virus latency.

A domain, previously termed RE1, exists within the herpes simplex virus type 1 genome potentially influencing expression of immediate early genes and the latency associated transcripts. This domain consists of 10 tandem copies of a CT-rich sequence. We demonstrate that this domain binds multiple host-cell factors that may allow RE1 to act either as a transcriptional regulator and/or to affect nucleosomal and DNA structure in the latent genome.

Two B cell subpopulations have distinct recirculation characteristics.

This report describes two subpopulations of B cells in sheep. These subpopulations have distinct recirculation characteristics and tissue distributions. Phenotypically the populations are distinguished by their differential expression of the complement receptors, CD21 (CR2) and CD11b/CD18 (CR3). CD11b+ B cells are surface (s)IgMhi, co-express CD11c but are L-selectin negative. They populate the splenic marginal zone but are absent from splenic and ileal Peyer's patch (IPP) follicles and both afferent and efferent lymph compartments. Fluorescent tracing experiments showed that the CD11b+ B cells are non-recirculating as they did not appear in lymph after intravenous inoculation but are restricted to the blood and spleen. The CD11b-negative population expresses a conformational determinant of CD21 that is recognized by the monoclonal antibody Du 2-74. These cells are sIgMlo and co-express L-selectin. They populate the splenic and IPP follicles, are absent from the splenic marginal zone and are the only B cells in afferent lymph, efferent lymph and all lymph nodes. Fluorescence tracing experiments showed that the CD21 B cells are recirculating cells with their entry into efferent lymph being detectable by 16 h and peaking at 24-30 h. These data suggest that there are at least two lineages of B cells in the sheep with different phenotypic, functional and recirculation characteristics.

Expression of the SU glycoprotein of maedi visna virus in baculovirus.

The envelope glycoprotein, gp 135, of the ovine lentivirus maedi visna virus is the main target for a specific antibody response in vivo, however, little is known about the specific regions of gp 135 which elicit this response. Research on the function of gp 135 has been hampered by the lack of reagents to study such structure/function relationships. We have used a baculovirus expression system to express gp 135 lacking the viral signal sequence. This recombinant protein is glycosylated and recognised by immune sera from clinically affected animals.

Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis.

Jaagsiekte retrovirus (JSRV) is an exogenous type D-related retrovirus specifically associated with a contagious lung cancer of sheep (sheep pulmonary adenomatosis; SPA). Recently, epithelial tumour cells in the lungs of SPA-affected sheep were identified as major sites of JSRV replication by immunological techniques and RT-PCR amplification of part of JSRV gag. JSRV was not detected outside the lungs and their draining lymph nodes. However, low levels of JSRV expression in non-respiratory tissues could have been masked by co-amplification of endogenous JSRV-related sequences, which were differentiated from JSRV by the lack of a Scal restriction site in the PCR product. To further investigate the pathogenesis of SPA, an exogenous virus-specific hemi-nested PCR was developed utilizing primers in the U3 region of JSRV LTR, where major differences between endogenous and exogenous sequences exist. This technique was shown to be > or = 10(5)-fold more sensitive than the previous gag PCR/ScaI digestion method. Using this new assay the tissue distribution of JSRV in sheep with natural and experimentally induced SPA was analysed. Proviral DNA and JSRV transcripts were found in all tumours and lung secretions of SPA-affected sheep (n = 22) and in several lymphoid tissues. The mediastinal lymph nodes draining the lungs were consistently demonstrated to be infected by JSRV (10/10). JSRV transcripts were also detected in spleen (7/9), thymus (2/4), bone marrow (4/8) and peripheral blood mononuclear cells (3/7). Proviral DNA was also detected in these tissues although in a much lower proportion of cases. JSRV was not detected in 27 samples from unaffected control animals (n = 15).

PCR-based detection and partial characterization of a retrovirus associated with contagious intranasal tumors of sheep and goats.

A type D-related retrovirus has been demonstrated in enzootic nasal tumors (ENTs) of sheep and goats. This retrovirus, ENT virus (ENTV), has antigenic cross-reactivity with the jaagsiekte sheep retrovirus (JSRV), which is associated with a contagious lung tumor of sheep (sheep pulmonary adenomatosis). Here, we present the first report of nucleic acid sequence from ENTV which confirms, at the nucleic acid level, that this retrovirus is related to JSRV yet apparently distinct from it. Reverse transcription-PCR followed by restriction enzyme digestion specifically identified ENTV. By this technique, ENTV was demonstrated exclusively in tumor tissues and exudates of animals with ENT. Thus, there is a unique and consistent association between ENT and the retrovirus, just as there is between JSRV and sheep pulmonary adenomatosis. This gives further weight to the hypothesis that these retroviruses are the etiologic agents of the tumors.

Identification of the sheep homologue of the monocyte cell surface molecule--CD14.

An ovine monocyte/macrophage cell surface antigen was recognized by three mouse monoclonal antibodies (mAbs) VPM65, VPM66 and VPM67. These mAbs also reacted with bovine cells. The antibodies immunoprecipitated a single, glycosyl-phosphatidylinositol-linked polypeptide of M(r) 55,000 which, when deglycosylated, was reduced to M(r) 53,000. They reacted strongly with peripheral blood monocytes, alveolar macrophages and peripheral blood granulocytes, and weakly with afferent lymph dendritic cells. They also reacted with macrophages in many different tissues but were non-reactive with lymphocytes. Competitive flow cytometry shows that these three mAbs recognize the same or a closely related epitope of a single antigen. An antigen-specific capture ELISA using the anti-human CD14 mAb (TUK4) revealed that all four mAbs associate with the same antigen. These data demonstrate that the mAbs react with the ovine homologue of the lipopolysaccharide (LPS)-LPS binding protein receptor, CD14.

The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep.

Sheep pulmonary adenomatosis ([SPA] ovine pulmonary carcinoma) is a transmissible lung cancer of sheep that has been associated etiologically with a type D- and B-related retrovirus (jaagsiekte retrovirus (JSRV]). To date it has been impossible to cultivate JSRV in vitro and therefore to demonstrate the etiology of SPA by a classical approach. In addition, the presence of 15 to 20 copies of endogenous JSRV-related sequences (enJSRV) has hampered studies at the molecular level. The aim of this study was to investigate whether the expression of exogenous JSRV was specifically associated with neoplasia in SPA-affected animals. Initially, we found that enJSRVs were transcribed in a wide variety of normal sheep tissues. Then, by sequencing part of the gag gene of enJSRV we established a ScaI restriction site in gag as a molecular marker for the exogenous form of JSRV. Restriction enzyme digestion of PCR products obtained from the amplification of cDNA from a total of 65 tissues collected from SPA-affected and unaffected control sheep revealed that the exogenous form of JSRV was exclusively and consistently present in tumor tissues and lung secretions of the affected animals. In addition, exogenous JSRV provirus was detected only in DNA from SPA tumors and not from nontumor tissues of the same animals. This study has demonstrated clearly that the exogenous form of JSRV is specifically associated with SPA tumors.

Epithelial tumour cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus.

Sheep pulmonary adenomatosis (SPA) is a naturally occurring contagious lung tumour of sheep which has been associated aetiologically with a type D- and B-related retrovirus (Jaagsiekte retrovirus; JSRV). To improve understanding of the aetio-pathogenesis of SPA, the distribution and the sites of JSRV replication in sheep with naturally or experimentally induced SPA or in unaffected controls were identified. New immunological reagents were produced and a blocking enzyme-linked immunosorbent assay (B-ELISA) and an immunohistochemical technique for the detection of JSRV major capsid protein at the tissue and cellular levels were developed. JSRV was detected only in the respiratory tract of sheep affected by pulmonary adenomatosis and specifically in the transformed epithelial cells of the alveoli of SPA-affected sheep.

Biochemical and phenotypic characterization of the ovine beta 2 (leucocyte) integrins.

This paper is concerned with the relationship of the three distinct members of the ovine beta 2-integrin family of leucocyte adhesion molecules that play an important role in cell-cell and cell-matrix interactions. A panel of monoclonal antibodies (mAbs) specific for sheep and cattle macrophages was characterized by flow cytometry, immunohistology, and immunoprecipitation for reactivity to the beta 2 integrins. Immunoprecipitation analysis of sheep antigens showed that these monoclonal antibodies could be divided into four distinct groups. All precipitated an M(r) 95,000 beta chain but they differed in the size or number (or both) of the alpha chains recognized. Group 1 precipitated alpha chains of M(r) 180,000; group 2 had M(r) 170,000 alpha chains; the group 3 alpha chain was of M(r) 150,000 and group 4 mAbs precipitated alpha chains of all three sizes. The relationship between these antibodies was demonstrated by sequential immunoprecipitation, which showed that the reactivities of antibodies in groups 1, 2 and 3 were mutually exclusive but that group 4 antibodies shared a common specificity with the other three groups. By analogy with the human and murine beta 2 integrin families, group 1 antibodies seemed to be specific for CD11a (LFA-1); group 2 were CD11b (CR3 or Mac1); group 3 were CD11c (CR4 or p150/95) and group 4 were CD18. In addition to different molecular weights, these antibodies had different cellular and tissue distributions. CD11a and CD18 were distributed identically. The antigens recognized by both were present on all the leucocyte populations. The mAbs recognizing CD11b reacted with a sub-population of peripheral blood B lymphocytes and all myeloid cells (alveolar macrophages, peripheral blood monocytes and granulocytes) except afferent lymph dendritic cells (ADC). Anti-CD11c (p 150/95 or CR4) antibodies reacted strongly with macrophages and ADC but were weakly reactive on monocytes and negative on neutrophils. CD11c was also present on a sub-population of peripheral blood B cells.