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Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRV(JS7)) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRV(JS7) is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.

Sheep pulmonary adenomatosis (SPA), also known as jaagsiekte or ovine pulmonary carcinoma, is a contagious lung cancer of sheep, originating from type II pneumocytes and Clara cells. Previous studies have implicated a type D retrovirus (jaagsiekte sheep retrovirus [JSRV]) as the causative agent of SPA. We recently isolated a proviral clone of JSRV from an animal with a spontaneous case of SPA (JSRV(21)) and showed that it harbors an infectious and oncogenic virus. This demonstrated that JSRV is necessary and sufficient to induce SPA. A major impediment in research on JSRV has been the lack of an in vitro tissue culture system for the virus. The experiments reported here show the first successful in vitro infection with this virus, using the JSRV(21) clone. JSRV(21) virus was obtained by transiently transfecting human 293T cells with a plasmid containing the JSRV(21) provirus driven by the human cytomegalovirus immediate-early promoter. Virus produced in this manner exhibited reverse transcriptase (RT) activity that banded at 1.15 g/ml in sucrose density gradients. Infection of concentrated JSRV(21) into ovine choroid plexus (CP), testes (OAT-T3), turbinate (FLT), and intestinal carcinoma (ST6) cell lines resulted in establishment of infection as measured by PCR amplification. Evidence that this reflected genuine infection included the fact that heat inactivation of the virus eliminated it, the levels of viral DNA increased with passage of the infected cells, and the infected cells released active RT as measured by the sensitive product enhancement RT assay. The RT activity released from the infected cells banded at 1.15 g/ml, and JSRV(21) provirus was transmitted from infected cells to uninfected ones by cocultivation. However, the amount of virus released from infected cells was low. These results suggest that the JSRV receptor is present on many ovine cell types and that the observed restriction of JSRV expression in vivo to tumor cells might be controlled by factors other than the viral receptor. Finally we tagged the U3 of pJSRV(21) with the bacterial supF gene, an amber suppressor tRNA gene. The resulting clone, termed pJSRV(supF), is infectious in vitro. It may be a useful tool for future studies on viral DNA integration, since the normal sheep genome contains 15 to 20 copies of highly JSRV-related endogenous sequences that cross-react with many JSRV hybridization probes.

Sheep pulmonary adenomatosis (SPA) is a contagious and experimentally transmissible lung cancer of sheep resembling human bronchiolo-alveolar carcinoma. A type D retrovirus, known as jaagsiekte sheep retrovirus (JSRV), has been associated with the etiology of SPA, but its exact role in the induction of the tumor has not been clear due to the lack of (i) a tissue culture system for the propagation of JSRV and (ii) an infectious JSRV molecular clone. To investigate the role of JSRV in the etiology of SPA, we isolated a full-length JSRV proviral clone, pJSRV21, from a tumor genomic DNA library derived from a natural case of SPA. pJSRV21 was completely sequenced and showed open reading frames in agreement with those deduced for the original South African strain of JSRV. In vivo transfection of three newborn lambs by intratracheal inoculation with pJSRV21 DNA complexed with cationic lipids showed that pJSRV21 is an infectious molecular clone. Viral DNA was detected in the peripheral blood mononuclear cells (PBMCs) of the transfected animals by a highly sensitive JSRV-U3 heminested PCR at various time points ranging from 2 weeks to 6 months posttransfection. In addition, proviral DNA was detected in the PBMCs, lungs, and mediastinal lymph nodes of two lambs sacrificed 9 months posttransfection, but no macroscopic or histological SPA lesion was induced. We prepared JSRV particles by transient transfection of 293T cells with a JSRV construct (pCMV2JS21) in which the upstream U3 was replaced with the cytomegalovirus early promoter. Four newborn lambs were inoculated with JSRV21 particles produced in this manner, and two of them showed the classical signs of SPA 4 months postinfection. The resulting tumors were positive for JSRV DNA and protein. Thus, JSRV21 is an infectious and pathogenic molecular clone and is necessary and sufficient to induce sheep pulmonary adenomatosis.

A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease.

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus specifically associated with a contagious lung tumor of sheep, sheep pulmonary adenomatosis (SPA). JSRV replicates actively in the transformed epithelial cells of the lung, and JSRV DNA and RNA have been detected in lymphoid tissues of naturally affected animals. To determine the lymphoid target cells of JSRV, CD4(+) T cells, CD8(+) T cells, B lymphocytes, and adherent cell (macrophage/monocyte) populations were isolated from the mediastinal lymph nodes of naturally affected sheep and lambs inoculated with JSRV. Cells were enriched to high purity and then analyzed for JSRV proviral DNA by heminested PCR, and the proviral burden was quantitated by limiting dilution analysis. JSRV proviral DNA was found in all subsets examined but not in appropriate negative controls. In sheep naturally affected with SPA, JSRV proviral burden was greatest in the adherent cell population. In the nonadherent lymphocyte population, surface immunoglobulin-positive B cells contained the greatest proviral burden, while CD4(+) and CD8(+) T cells contained the lowest levels of JSRV proviral DNA. In most of the cases (5 of 8), provirus also could be detected in the peripheral blood mononuclear cell (PBMC) population. A kinetic study of JSRV infection in the mediastinal lymphocyte population of newborn lambs inoculated with JSRV found that JSRV proviral DNA could be detected as early as 7 days postinoculation before the onset of pulmonary adenomatosis, although the proviral burden was greatly reduced compared to adult natural cases. This was reflected in the levels found in PBMC since proviral DNA was detected in 2 of 13 animals. At the early time points studied (7 to 28 days postinoculation) no one subset was preferentially infected. These data indicate that JSRV can infect lymphoid and phagocytic mononuclear cells of sheep and that dissemination precedes tumor formation. Infection of lymphoid tissue, therefore, may play an important role in the pathogenesis of SPA.

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.