Jaagsiekte sheep retrovirus is present at high concentration in lung fluid produced by ovine pulmonary adenocarcinoma-affected sheep and can survive for several weeks at ambient temperatures.

Jaagsiekte sheep retrovirus (JSRV) causes a fatal lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). OPA is a significant disease in many sheep-rearing countries and there is no effective method of control. A unique feature of OPA is the overproduction of fluid in the lung of affected animals. This lung fluid contains JSRV and provides a means of transmission through the inhalation of virus. In this study we demonstrated that lung fluid from different OPA cases contained between 10(7) and 10(10) copies of JSRV RNA per ml. Examination of JSRV RNA survival under conditions that mimic natural conditions suggested that intact JSRV virions may persist for several weeks in the environment. These are the first quantitative data on JSRV in lung fluid and provide valuable information for implementing appropriate biosecurity measures to control the spread of JSRV in the field.

Expression of the jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5' LTR) and the env region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.

Multiclonal pattern of Jaagsiekte sheep retrovirus integration sites in ovine pulmonary adenocarcinoma.

Insertional mutagenesis and envelope (Env)-mediated oncogenesis are hypothesized mechanisms by which Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA). Twenty-eight JSRV integration sites in lung tumors (LTs) from four sheep with OPA were cloned and sequenced by a multiple step gene walking technique. Using nested PCR, clonal expansion of these integration sites could be detected, if at all, only in the localized regions of LT from which the integration sites were derived. One sheep had a viral integration site in a sequence with 85 and 81% identity, respectively, over 100 bp to exon 2 of the human and mouse receptor protein tyrosine phosphatase gamma genes. Clonal integration of Jaagsiekte sheep retrovirus in this gene was demonstrated by nested PCR and Southern blot hybridization in the DNA sample from which the integration site was cloned, but not in other LT or kidney DNA samples from the same sheep. OPA may develop from multiple independent oncogenic events and a role for insertional mutagenesis cannot be ruled out.

Jaagsiekte sheep retrovirus-specific immune responses induced by vaccination: a comparison of immunisation strategies.

Jaagsiekte sheep retrovirus (JSRV) is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). No JSRV-specific immunological responses have been detected in clinical cases of OPA or in experimentally infected lambs. The aim of the present study was to induce immune responses in sheep against JSRV proteins using several immunisation strategies. The vaccines were administered subcutaneously and intradermally, or intranasally, in adjuvant. Antibodies were measured by ELISA and immunoblotting, and T cell responses by lymphoproliferation assay. Antibodies specific for JSRV-capsid protein were induced by inoculation of recombinant proteins in adjuvant, and transient JSRV-specific T cell responses by intranasal inoculation with inactivated virus. These results will help in the design of a protective vaccine against JSRV infection and the development of OPA.

A PCR technique for the detection of Jaagsiekte sheep retrovirus in the blood suitable for the screening of ovine pulmonary adenocarcinoma in field conditions.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring contagious lung neoplasia caused by jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, JSRV proviral DNA sequences can be found in peripheral blood leukocytes (PBLs) in clinically affected and in a proportion of in contact animals. In this study, existing hemi-nested PCR procedure is compared with a new one-step PCR technique that was developed to minimise potential DNA contamination and reduce sample and reagent handling. Different blood preparations were assessed and the best results were achieved on DNA prepared from buffy coat. The sensitivity of this PCR was lower in JSRV infected sheep without lesions of OPA than in clinically affected sheep, which indicate that this PCR may not be not fully appropriate for screening of individual sheep, but rather to provide results at flock level. This PCR is the only currently available blood test for detection of JSRV infected sheep and may be useful in epidemiological studies and in control programmes of OPA.

Successful induction of ovine pulmonary adenocarcinoma in lambs of different ages and detection of viraemia during the preclinical period.

Ovine pulmonary adenocarcinoma (OPA) can be reproduced consistently in neonatal lambs by intratracheal injection of inocula containing jaagsiekte sheep retrovirus (JSRV). In this study, clinical disease, confirmed pathologically as OPA, was induced in a high proportion of lambs that had been inoculated intratracheally with infectious lung fluid at 1, 3 and 6 months of age. The incubation periods, however, were longer in these three age groups than in 1-week-old lambs that were used as controls. Viraemia was detected in all age groups before onset of clinical signs, but occurred later in older animals. These results suggest an age-dependent susceptibility to OPA that could be determined by the availability of JSRV target cells in the ovine lung. The feasibility of inducing OPA in older lambs and detecting JSRV viraemia in preclinical stages enables improved studies on the pathogenesis, assessment of vaccines, diagnosis and control of the disease.

Analysis of integration sites of Jaagsiekte sheep retrovirus in ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumor of sheep caused by Jaagsiekte sheep retrovirus (JSRV). To test the hypothesis that JSRV insertional mutagenesis is involved in the oncogenesis of OPA, we cloned and characterized 70 independent integration sites from 23 cases of OPA. Multiple integration sites were identified in most tumors. BLAST analysis of the sequences did not disclose any potential oncogenic motifs or any identical integration sites in different tumors. Thirty-seven of the integration sites were mapped to individual chromosomes by PCR with a panel of sheep-hamster hybrid cell lines. Integration sites were found on 20 of the 28 sheep chromosomes, suggesting a random distribution. However, four integration sites from four different tumors mapped to chromosome 16. By Southern blot hybridization, probes derived from two of these sites mapped to within 5 kb of each other on normal sheep DNA. These sites were found within a single sheep bacterial artificial chromosome clone and were further mapped to only 2.5 kb apart, within an uncharacterized predicted gene and less than 200 kb from a mitogen-activated protein kinase-encoding gene. These findings suggest that there is at least one common integration site for JSRV in OPA and add weight to the hypothesis that insertional mutagenesis is involved in the development of this tumor.

Characterization of enzootic nasal tumour virus of goats: complete sequence and tissue distribution.

The complete genome sequence of a new isolate of enzootic nasal tumour virus (ENTV-2), associated with enzootic nasal adenocarcinoma (ENA) of goats, was determined. The genome exhibits a genetic organization characteristic of beta-retroviruses. ENTV-2 is closely related to the retrovirus (ENTV-1) associated with enzootic adenocarcinoma of sheep, and to jaagsiekte retrovirus. The main sequence differences between these viruses reside in orfX, the U3 LTR, two small regions in gag and the transmembrane (TM) region of env. Sequence analysis of the TM region of env from several sheep and goats naturally affected by ENA suggested that ENTV-1 and ENTV-2 are distinct viruses rather than geographical variants. Although both viruses transform secretory epithelial cells of the ethmoid turbinate, the study of their tissue distribution using specific PCRs showed that ENTV-2 establishes a disseminated lymphoid infection whereas ENTV-1 is mainly confined to the tumour.