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. 1999 Aug;73(8):6964-72.
doi: 10.1128/JVI.73.8.6964-6972.1999.

Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep

M Palmarini  1 , J M Sharp, M de las Heras, H Fan
Affiliations

Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep

M Palmarini et al. J Virol. 1999 Aug.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Cloning of JSRV21. The strategy used for the isolation of λJSRV21 is shown.
FIG. 2
FIG. 2
JSRV21-based plasmid constructs and in vitro synthesis of viral particles. (a) Schematic representation of the genomic organization of the JSRV21 provirus; standard retroviral notation is used. The proviral genome is typical of type B and type D retroviruses, with pro in a different open reading frame from pol. Note the presence of an accessory open reading frame (orf-x) overlapping pol. (b) pJSRV21 and pCMV2JS21 plasmid constructs. In pCMV2JS21, the U3 region of the proximal LTR was replaced by the human CMV promoter. (c) Western blot of 300-fold-concentrated supernatant from 293T cells transiently transfected with pCMV2JS21 and collected 24, 48, and 72 h posttransfection. The filters were probed with a rabbit polyclonal antiserum against the major capsid protein (CA) of JSRV (28). Lung secretions collected from an SPA-affected animal and concentrated in the same way as the 293T supernatant were used as a positive control (LF). Concentrated supernatant from mock-transfected 293T cells was used as a negative control (M). The 26-kDa CA protein is indicated.
FIG. 3
FIG. 3
Buoyant-density analysis of JSRV21. (a) Intact JSRV21 particles prepared by transient transfection of 293T cells with pCMV2JS21 were analyzed by isopycnic centrifugation in a 25 to 60% (wt/wt) sucrose gradient. Adjacent fractions were pooled, and exogenous RT activity was determined (solid lines). The densities of each fraction are shown in grams per milliliter (dashed lines). (b) JSRV21 was treated with 0.1% Triton X-100 and analyzed as in panel a.
FIG. 4
FIG. 4
Induction of SPA in JSRV21-infected lambs. (A to C) Lung tumor tissues from JSRV21-infected lambs were fixed in neutral 10% formalin, embedded in paraffin, and sectioned by routine procedures. Hematoxylin and eosin-stained lung tumor sections are shown. (A) Low-magnification micrograph (magnification, ×23; bar, 380 μm) showing many neoplastic foci in the microscopic field (some are indicated by arrows). (B) High-magnification micrograph (magnification, ×372; bar, 40 μm) of a neoplastic nodule with a clear papillary pattern (∗). Myxoid tissue containing cells with elongated or round nuclei is present in the interstitium of the neoplastic tissue. (C) Papillary proliferation (∗) occluding the lumen of a bronchiolus. Magnification, ×372; bar, 40 μm. (D to E) Immunohistochemistry for JSRV CA antigen developed with an avidin-biotin peroxidase complex kit (ABC; Vector Laboratories). Carazzi’s hematoxylin alone was used as counterstain. Magnification, ×372; bar, 40 μm. (D) A neoplastic focus (∗) is shown where most of the neoplastic cells have a brownish cytoplasmatic stain indicative of JSRV CA antigen after staining with a rabbit anti-CA antiserum as primary antibody. No staining is present in the cells infiltrating the tumor or in adjacent normal cells. (E) Rabbit preimmune serum was used as the primary antibody in lung tumor sections in parallel to those in panel D; no brown staining was visible in the tumor nodule (∗). (F) A lung section from an uninoculated lamb was tested for JSRV CA antigen under conditions similar to those in panel D; there were no antigen-positive cells.
FIG. 5
FIG. 5
Exogenous viral DNA in JSRV21-induced tumors. (A) Lung DNA (500 ng) from four lambs (lanes 1 to 4) inoculated with JSRV21 was tested for exogenous virus sequences by PCR amplification with primers from the U3 region of the LTR that are specific for exogenous JSRV (30). Three of four inoculated lambs showed evidence of exogenous JSRV21 DNA in lung tissue, including the two with SPA tumors (lanes 1 and 2). As expected, no PCR product was evident in amplifications of lung DNA from the uninoculated control lambs (lanes 5 and 6). Other controls included distilled water in the PCR amplification (lane −) and pJSRV21 plasmid DNA in the amplification (lane +). (B) The presence of the exogenous virus-specific ScaI restriction site in gag was tested in the tumor DNAs of lambs 1 and 2. Lung tumor or normal lung DNA (500 ng) was subjected to LTR-gag hn-PCR followed by digestion with ScaI as described previously (27). The products were then analyzed by agarose gel electrophoresis. U, uncut PCR product; C, PCR product cut with ScaI. Both lambs 1 and 2 showed the ScaI restriction site specific for the exogenous JSRV sequences; lambs 5 and 6 showed no PCR products. PCR controls included distilled water (lanes −) and pJSRV21 plasmid DNA (lanes +). Molecular weight markers are indicated on the side of both panels (in base pairs).
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