Detection of Jaagsiekte sheep retrovirus in the peripheral blood during the pre-clinical period of ovine pulmonary adenomatosis.

The envelope protein (Env) of the Jaagsiekte sheep retrovirus (JSRV) is known to be a unique oncoprotein responsible for inducing ovine pulmonary adenocarcinoma (OPA). The objective of this study was to prepare a specific monoclonal antibody (mAb) against the JSRV Env protein using bioinformatic analysis. According to the structure and epitope prediction results of JSRV Env, the JSRV-Env572-615 antigen was prepared via peptide synthesis (amino acid sequence 572-615, denoted as JSRV-Env572-615). BALB/c mice were immunized to prepare the anti-JSRV-Env572-615 mAb. Spleen cells were fused with SP2/0 myeloma cells after being screened by indirect ELISA and cloned by limiting dilution. The specificity of mAb was evaluated by western blot analysis and immunohistochemistry assays. Western blot results showed that the JSRV Env protein was able to bind to mAb with high specificity. Immunohistochemistry assays demonstrated that the mAb was able to recognize JSRV Env in adenomatous hyperplasia of the lung. Furthermore, JSRV was detected in peripheral blood leukocytes during the pre-clinical period of OPA in 2 of the 25 sheep using this newly synthesized mAb. Therefore, this mAb may be a useful tool for the detection of JSRV in sheep.

IFITM proteins restrict viral membrane hemifusion.

The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.

Microsatellites in immune-relevant regions and their associations with Maedi-Visna and ovine pulmonary adenocarcinoma viral diseases.

Maedi-Visna (MV) and ovine pulmonary adenocarcinoma (OPA) are two retroviral diseases occurring worldwide that affect adult sheep. Differences in incidence, which may be related to sheep-rearing and housing choices, as well as to genetics, and disease progression have been reported for both diseases. In this work four microsatellites located in immune-relevant regions, the major histocompatibility complex (MHC) region, interferon-γ and interleukin-12p35, were genotyped to determine their association with disease progression. The analysed sample included Latxa sheep with and without OPA and MV-characteristic lesions in their lungs. The microsatellites in the MHC were the most diverse, while the ones located in the cytokines were the less polymorphic. In the case of IFN-γ the results suggested the presence of null alleles. Significant results were detected for several microsatellite alleles in the association analysis carried out by logistic regression. All statistical analyses included a flock effect adjustment to avoid false positives due to genetic structuration. MHC Class I microsatellite alleles OMHC1*205 and OMHC1*193 were associated with disease progression for Maedi and OPA, respectively. Moreover, MHC Class II microsatellite allele DRB2*275 was associated with presence of lesions in Maedi. Furthermore, the MHC microsatellites were combined for a bioinformatic haplotype inference with the PHASE software. In total, 73 haplotypes were detected, 18 of them in more than 6 animals. After standard and weighted logistic regression analysis, two of them were significantly associated with susceptibility: OMHC1*205-DRB2*271 for Maedi and OMHC1*193-DRB2*271 for OPA, both with the Class I microsatellite alleles associated in the marker by marker study. Although more extensive analyses are needed to disentangle the relationship between host genetics and disease, as far as we know this is the first study demonstrating a significant association between sheep MHC Class I microsatellite alleles and susceptibility to Maedi-Visna and OPA viral diseases.

MHC class II DRB1 gene polymorphism in the pathogenesis of Maedi-Visna and pulmonary adenocarcinoma viral diseases in sheep.

Ovine pulmonary adenocarcinoma (OPA) and Maedi-Visna (Maedi) are two chronic respiratory diseases of retroviral origin which occur worldwide. It is known that different host genetic factors influence the outcome of viral infections. To determine if variation in the Mhc-DRB1 gene was associated with progression to these ovine diseases, sheep lungs with and without OPA and Maedi lesions were collected. A sequence-based method was applied and 40 different alleles were detected in the sample analysed. In the allele-by-allele association analysis, allele DRB1*0325 had a significant association with susceptibility to Maedi (P = 0.045). For OPA, DRB1*0143 and DRB1*0323 were significantly associated with susceptibility (P = 0.024 and P = 0.029), and allele DRB1*0702 was significantly associated with resistance (P = 0.012). Based on these results, the Mhc-DRB1 alleles were classified by effect in three categories-susceptible (S), resistant (R) and neutral (N)-and animals were reassigned the genotypes as S/S, S/R, S/N, R/R, R/N and N/N. In a second analysis, penalised logistic regression models including a flock effect were run. In Maedi, significant association was detected for the N/S heterozygote (P = 0.0007), but not for the S/S homozygote, probably as a result of the low number of S/S animals. In OPA, association was detected for both the S/S and R/R homozygotes (P = 0.005 and P = 0.047). This allele grouping method may be applied in association studies with highly variable genes. This is the first study demonstrating significant associations between sheep Mhc-DRB1 alleles and susceptibility to OPA and Maedi. Therefore, both diseases are suitable candidates for more comprehensive genetic studies.

An influx of macrophages is the predominant local immune response in ovine pulmonary adenocarcinoma.

Infection with a retrovirus, Jaagsiekte sheep retrovirus (JSRV), causes ovine pulmonary adenocarcinoma (OPA). The excess production of surfactant proteins by alveolar tumour cells results in increased production of pulmonary fluid, which is characteristically expelled through the nostrils of affected sheep. The immune response to JSRV and the tumour is poorly understood: no JSRV-specific circulating antibodies or T cells have been detected to date. The aim of the present study was to obtain phenotypic evidence for a local immune response in OPA lungs. Specific-pathogen free lambs were infected intratracheally with JSRV. When clinical signs of OPA were apparent, the lungs were removed at necropsy and immunohistochemistry (IHC) was performed on lung sections using a panel of mouse anti-sheep mAbs. No influx of dendritic cells, B cells, CD4, CD8 or gammadelta T cells was seen in the neoplastic nodules or in their periphery. MHC Class II-positive cells were found intratumourally, peritumourally and in the surrounding alveolar lumina. In the tumours, many of these cells were shown to be fibroblasts and the remainder were likely to be mature macrophages. In the alveolar lumen, the MHC Class II-positive cells were CD14-positive and expressed high levels of IFN-gamma. They appeared to be immature monocytes or macrophages which then differentiated to become CD14-negative as they reached the periphery of the tumours. A high level of MHC Class I expression was detected on a range of cells in the OPA lungs but the tumour nodules themselves contained no MHC Class I-positive cells. On the basis of these findings, it is proposed that the lack of an effective immune response in OPA could result from a mechanism of peripheral tolerance in which the activity of the invading macrophages is suppressed by the local environment, possibly as a consequence of the inhibitory properties of the surfactant proteins.

Natural history of JSRV in sheep.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour of sheep and, rarely, goats that arises from two types of secretory epithelial cell that retain their luxury function of surfactant synthesis and secretion. It is classified as a low-grade adenocarcinoma and is viewed as a good model for epithelial neoplasia because of its morphological resemblance to the human lung tumour, bronchioloalveolar adenocarcinoma. OPA is present in most of the sheep rearing areas of the globe and, in affected flocks, tumours are present in a high proportion of sheep. OPA is associated with the ovine retrovirus, jaagsiekte sheep retrovirus (JSRV), and is transmissible only with inocula that contain JSRV. All sheep contain JSRV-related endogenous viruses, but JSRV is an exogenous virus that is associated exclusively with OPA. JSRV is detected consistently in the lung fluid, tumour and lymphoid tissues of sheep affected by both natural and experimental OPA or unaffected in-contact flockmates and never in sheep from unaffected flocks with no history of the tumour. JSRV replicates principally in the epithelial tumour cells, but also establishes a disseminated infection of several lymphoid cell types, including peripheral blood leukocytes (PBLs). Longitudinal studies in flocks with endemic OPA have revealed JSRV in PBLs before the onset of clinical OPA and even in the absence of discernible lung tumour. The prevalence of JSRV infection is 40%-80%, although only 30% of sheep appear to develop OPA lesions. A unique feature of OPA is the absence of a specific humoral immune response to JSRV, despite the highly productive infection in the lungs and the disseminated lymphoid infection. This feature is associated with reduced responsiveness to some mitogens, although the phenotypic profile of the peripheral blood remains unaltered. The reduced response is an early and sustained event during infection and may indicate that the failure of infected sheep to produce specific antibodies to JSRV is a direct consequence of infection.

Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma.

Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). To monitor changes in cellular immune function during JSRV infection, lymphoproliferation in response to various mitogens was measured in the blood of conventionally housed and specific-pathogen-free lambs experimentally infected with JSRV until the development of OPA and compared with uninfected control lambs. In addition, blood samples collected from adult field cases in the terminal stages of OPA and control adult sheep were compared. No difference in the proliferative response to phytohaemagglutinin and pokeweed mitogen between the animal groups was detected. In contrast, reduced responses to concanavalin A stimulation were demonstrated in the JSRV-inoculated lambs, prior to the onset of clinical disease, and also in the terminally ill adult sheep. Peripheral blood leukocytes were monitored to identify phenotypic frequency alterations. The CD4 lymphocytopaenia and neutrophilia reported previously in adult OPA cases were demonstrated but similar phenotypic changes were not identified during experimental infection.

Sheep pulmonary adenomatosis: characterization of two pathological forms associated with jaagsiekte retrovirus.

Pathological and immunohistochemical studies were performed on the lungs of 10 sheep with lesions of "classical" sheep pulmonary adenomatosis (SPA) and six sheep with "atypical" lung tumours. Lung tumour samples and other tissues from the same 16 animals were tested for the presence of jaagsiekte retrovirus (JSRV) by a polymerase chain reaction (PCR) that amplified a portion of the U3 long terminal repeat. The differences in the gross appearance of the classical and atypical forms paralleled the histopathological differences. The latter mainly concerned the stroma of the tumours which in the atypical cases was more heavily infiltrated by inflammatory cells and connective tissue fibres. JSRV major capsid protein was detected immunohistochemically in the epithelial transformed cells of both classical and atypical tumours, but the immune reactivity was slightly milder in atypical SPA. Proviral U3 sequences of JSRV were detected by specific PCR in all the tumour samples. Furthermore, the sequences of amplimers obtained from the two different pathological forms of the tumour were very similar. However, the dissemination of JSRV to other organs was greater in sheep with classical SPA than in those with atypical SPA. The pathological and virological features of these two forms of tumour are compared in an attempt to clarify whether classical and atypical SPA are two separate diseases or different expressions of a single disease spectrum.

Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis.

Enzootic nasal tumour (ENT) and sheep pulmonary adenomatosis (SPA) are two contagious adenocarcinomas of the respiratory tract of sheep and goats. Both diseases are associated with related, but distinct, type-D-retroviruses (ENTV and JSRV respectively). No evidence of circulating antibodies has been described in animals affected by either ENT or SPA using antigens from natural sources. We evaluated the usefulness of a recombinant JSRV capsid protein (JSRV-CA) as antigen to study the antibody responses of animals naturally affected by ENT or SPA, using immunoblotting. Positive reactions were detected in the sera of both affected and unaffected sheep and goats. The reactivity was abolished completely by absorption with the GST fusion partner but not by JSRV-CA, suggesting that it was not specific. The results support prior observations indicating that sheep and goats infected by JSRV and ENTV do not develop specific humoral responses to these retroviruses.

Production of a macrophage chemotactic factor by cultured jaagsiekte tumour cells.

The increase of alveolar macrophages in jaagsiekte sheep lungs is not caused by excessive surfactant production but is due to a chemotactic factor secreted by the tumor cells. This factor has a molecular mass in the region of 13 kilodaltons, is stable at 56 degrees C but labile at 100 degrees C and, being sensitive to proteases, indicates that it is a small protein molecule.

[Morphofunctional characterization of the immune system in ovine adenomatosis]. / Morfofunktsional'noe sostoianie immunnoi sistemy pri adenomatoze ovets.

In sheep with adenomatosis, enhanced activity is exhibited by both the central and peripheral organs of immunity. Histologic manifestations of this in the thymus include blast transformation of the T and B zones, numerous mitoses, elevated numbers of unicellular thymus corpuscles, and accumulations of thymocytes around these corpuscles and capillaries, while in the lymph nodes and spleen they include many mitoses in the reactive centers of lymphatic follicles and in the mentle and marginal zones, and increased numbers of lymphocytes, neutrophils, plasma cells, and PAS-positive cells. The intensified functioning of lymphoid tissues combines with very pronounced phagocytic reactions in the immune organs and pulmonary parenchyma and with adenomatous proliferation.

The localization of a Mason-Pfizer monkey virus-related antigen in jaagsiekte tumour tissue and cell lines.

Mason-Pfizer monkey virus-related antigen was detected in 3 out of 5 jaagsiekte lungs examined using a direct immunoperoxidase staining technique with anti-MPMV p27 serum. Most of the antigen was localized in the alveolar lumina of the lesions. The reaction was further characterised on immune blots and found to involve a protein with a molecular mass of 29 000 daltons (JSRV p29). JSRV p29 antigen was also detected in 2 jaagsiekte cell lines.

Serum and cerebrospinal fluid concentrations of immunoglobulin G in Suffolk sheep with scrapie.

Determinations were made by laser nephelometry of serum and CSF immunoglobulin (Ig) G concentrations in Suffolk sheep with naturally occurring scrapie. The serum IgG concentrations in 3 sheep with confirmed or suspected scrapie were between 2,140 and 3,290 mg of IgG/100 ml, and the CSF values were between less than 10 and 75 mg of IgG/100 ml. In 8 clinically healthy (control) sheep, serum IgG concentrations were 2,647 to 7,380 mg/100 ml and CSF IgG concentrations were between 0 (undetectable) and 162 mg/100 ml. A sheep with pulmonary adenomatosis had 1,445 mg of IgG/100 ml of serum. The results indicated that neither serum nor CSF IgG concentrations were increased in sheep with naturally occurring infection with scrapie and that the severity of the disease did not correspond with the IgG concentration.

Sheep pulmonary adenomatosis: demonstration of a protein which cross-reacts with the major core proteins of Mason-Pfizer monkey virus and mouse mammary tumour virus.

A retrovirus that causes pulmonary adenomatosis, a contagious lung tumour of sheep, contains a 25 000 mol. wt. polypeptide which cross-reacts with the major core protein (p27) of Mason-Pfizer monkey virus and mouse mammary tumour virus.

On the aetiology of Jaagsiekte.

A summary is given of the results obtained in experimental transmission of jaagsiekte by means transplantation of cell cultures. Evidence is also presented of transformation as the mechanism of oncogenesis and possibility of a viral aetiology is discussed briefly.

The serological relationship of herpesvirus ovis to other herpesviruses and its possible involvement in the aetiology of jaagsiekte.

Cross-neutralization studies showed that 3 different isolates of herpesvirus ovis from cell cultures derived from the lungs of sheep suffering from jaagsiekte were not only identical but were also related to similar isolate made in Scotland. No relationship, however, could be established between herpesvirus ovis and common bovine or equine herpesviruses. Antibodies to herpesvirus ovis were present in roughly 70% of all animals tested and no evidence was obtained for the involvement of the virus in the aetiology of jaagsiekte. On the other hand, the absence of antibodies in sheep sera from Iceland as well as the other data obtained in this study did not exclude involvement of the virus in jaagsiekte.

Lung carcinoma of sheep (jaagsiekte): tissue-bound precipitating antibodies.

Firmly bound IgG was examined and its activity was tested in spontaneous lung carcinoma of sheep (jaagsiekte). High levels of IgG were extracted by acid-glycine buffer (GB) at pH 2.8 from both the tumor tissue and normal lung, which were exhaustively prewashed by phosphate-buffered saline (PBS) at pH 7.4. The first acid-GB eluates of the normal or tumor tissue acted in the immunodiffusion test as precipitating antibodies against antigens of the first PBS eluate of the tumor tissue. In addition, free intracytoplasmic type A particles were observed in cell-free tumor extract.

Antigenic shift of visna virus in persistently infected sheep.

Visna viruses isolated from persistently infected sheep were antigenically distinct from the plaque-purified virus used for inoculation. The selection of antigenic variants under antibody pressure, thought to occur in vivo, was reproduced in sheep cell cultures inoculated with plaque-purified visna virus and maintained in antibody. Antigenic shift may be a mechanism for persistence of virus in slow or recurrent viral infections.

Characteristics of an ovine herpesvirus associated with pulmonary adenomatosis (jaagsiekte) in sheep.

The isolation of an ovine herpesvirus from a cell culture of an adenomatous sheep lung is reported, confirming previous observations of a possible association of a herpesvirus with this tumour. Some growth properties and morphological characteristics of the virus are described, as well as serological data supporting a possible relationship between tumour and virus. Attempts to produce jaagsiekte by intratracheal injection of virus into lambs were unsuccessful, suggesting that a second factor may be involved inthe oncogenic process possibly similar to that proposed for the well-known EBV-Burkitt's lymphoma system.