Christina Cousens

Nucleotide Sequence of EV1, a British Isolate Of Maedi-Visna Virus

We have isolated a maedi-visna-like virus from the peripheral blood mononuclear cells of a British sheep displaying symptoms of arthritis and pneumonia. After brief passage in fibroblasts this virus (designated EV1) was used to infect choroid plexus cells. cDNA clones of the virus were prepared from these cells and sequenced. Gaps between non-overlapping clones were filled using gene amplification by the polymerase chain reaction. The genome structure is similar to that described for visna virus strain 1514, and differs from that described for visna virus strain SA-OMVV in not having a W reading frame. Overall the genome differs by about 20% between each of these strains, but there is fivefold variation in the amount of divergence of derived amino acid sequences of different open reading frames. Two sequenced EV1 clones each contain only one copy of the 43 bp repeat, with paired AP-1 sites, which is a feature of other ruminant lentiviral long terminal repeats (LTRs). However, analysis of viral DNA in infected cells by gene amplification shows that LTRs with two repeats do occur, albeit at a relatively low frequency.

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).

Expression of the Jaagsiekte Sheep Retrovirus Envelope Glycoprotein Is Sufficient To Induce Lung Tumors in Sheep

ABSTRACT 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.

Enzootic Nasal Adenocarcinoma of Sheep and Goats

Enzootic nasal adenocarcinoma is a contagious tumour of the mucosal nasal glands affecting young adult sheep or goats. The disease occurs naturally in all continents except Australia and New Zealand. Clinical signs include continuous nasal discharge, respiratory distress, exophthalmos and skull deformations. The tumour is classified histologically as a low-grade adenocarcinoma. Nasal glands of both respiratory and olfactory muosal glands seem to be the origin of the neoplasia. It has been experimentally transmitted in sheep and goats using either tumour extracts or concentrated nasal fluids. Two distinct retroviruses are implicated in the aetiology of the neoplasia one in sheep (ONAV) and one in goats (CNAV). We suggest that jaagsiekte sheep retrovirus (JSRV), ONAV, CNAV, and their endogenous counterparts represent a unique family of retroviruses. The similarities between these viruses suggests that any control strategies, including vaccination, may be appropriate to both diseases. The differences, however, represent a unique resource for delineating the function of individual regions of the virus. It is intriguing that whilst ONAV and CNAV appear to be as different to each other as they are to JSRV, that they have very similar disease pathologies, distinct from that of OPA. Additionally, all three exogenous viruses manage to avoid instigating any apparent immune response. Whether this is indeed a result of tolerance induced by the endogenous counterparts or whether the viruses themselves have unique immunosuppressive properties will be an important finding.

Analysis of Integration Sites of Jaagsiekte Sheep Retrovirus in Ovine Pulmonary Adenocarcinoma

ABSTRACT 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.

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.

Cellular Differentiation and Proliferation in the Ovine Lung during Gestation and Early Postnatal Development

This study investigates epithelial cell differentiation and proliferation in specific anatomical regions of the ovine lung during prenatal and postnatal development. Immunohistochemistry was used to identify ciliated epithelial cells, Clara cells, neuroepithelial bodies and type II pneumocytes in the lungs of preterm (67, 127 and 140 days of gestation), full-term (147 days) and postnatal (9, 16 and 91 days old) lambs. Differentiation of ciliated epithelial cells was seen at 67 days of gestation and at term for Clara cells. Neuroepithelial bodies were first detected at 127 days of gestation. From 16 to 91 days of age there was a significant (P <0.05) increase in beta-tubulin (present in ciliated epithelial cells) and Clara cell protein (present in Clara cells) in multiple regions of the lung. Detection of Ki67, a marker of proliferation, in preterm lambs showed a reduction in proliferation index in multiple anatomical regions of the lung between 70 days of gestation and term. Cell proliferation increased following parturition, and then decreased between 16 and 91 days of age, with the largest reduction occurring in the alveolar compartment. Knowledge of which cells are present at specific times of lung development provides valuable information on the anatomy of the ovine lung, improving its use as a model for ovine and human neonatal disease. In addition, the antibodies used here will be valuable for future studies requiring the identification and quantification of respiratory epithelial cell phenotypes in the sheep lung.