Transcriptional Response of Ovine Lung to Infection with Jaagsiekte Sheep Retrovirus.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially expressed in JSRV-infected lung compared to noninfected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and reverse transcription-quantitative PCR. A key finding was the activation of anterior gradient 2, yes-associated protein 1, and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity, including genes encoding cytokines, chemokines, and complement system proteins. In contrast, there was little evidence for the upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with the transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma.IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.

First confirmation by PCR of Jaagsiekte sheep retrovirus in Ireland and prevalence of ovine pulmonary adenocarcinoma in adult sheep at slaughter.

BACKGROUND: Ovine pulmonary adenocarcinoma (OPA), caused by Jaagsiekte sheep retrovirus (JSRV), is characterised by the development of invariably fatal lung tumours primarily in adult sheep. High infection rates and disease prevalence can develop during initial infection of flocks, leading to on-farm economic losses and animal welfare issues in sheep with advanced disease. The disease has been reported in Ireland and is notifiable, but the presence of JSRV has never been confirmed using molecular methods in this country. Additionally, due to the difficulties in ante-mortem diagnosis (especially of latently-infected animals, or those in the very early stages of disease), accurate information regarding national prevalence and distribution is unavailable. This study aimed to confirm the presence of JSRV in Ireland and to obtain estimates regarding prevalence and distribution by means of an abattoir survey utilising gross examination, histopathology, JSRV-specific reverse transcriptase polymerase chain reaction (RT-PCR) and SU protein specific immunohistochemistry (IHC) to examine the lungs of adult sheep. RESULTS: Lungs from 1911 adult sheep were examined macroscopically in the abattoir and 369 were removed for further testing due to the presence of gross lesions of any kind. All 369 were subject to histopathology and RT-PCR, and 46 to IHC. Thirty-one lungs (31/1911, 1.6%) were positive for JSRV by RT-PCR and/or IHC but only ten cases of OPA were confirmed (10/1911, 0.5%) Four lung tumours not associated with JSRV were also identified. JSRV-positive sheep tended to cluster within the same flocks, and JSRV-positive sheep were identified in the counties of Donegal, Kerry, Kilkenny, Offaly, Tipperary, Waterford and Wicklow. CONCLUSIONS: The presence of JSRV has been confirmed in the Republic of Ireland for the first time using molecular methods (PCR) and IHC. In addition, an estimate of OPA prevalence in sheep at slaughter and information regarding distribution of JSRV infection has been obtained. The prevalence estimate appears similar to that of the United Kingdom (UK). Results also indicate that the virus has a diverse geographical distribution throughout Ireland. These data highlights the need for further research to establish national control and monitoring strategies.

Modulation of autophagy affected tumorigenesis induced by the envelope glycoprotein of JSRV.

Ovine pulmonary adenocarcinoma (OPA), caused by the jaagsiekte sheep retrovirus (JSRV), is a chronic, progressive, and contagious lung tumor that seriously affects sheep production. It also represents a valuable animal model for several human lung adenocarcinomas. However, little is known about the role of autophagy in OPA tumorigenesis. Here, Western blotting combined with transmission electron microscopy examination and Cyto-ID dye staining was employed for evaluation of changes of autophagic levels. The results of the present study showed that expression of the autophagy marker proteins Beclin-1 and LC3 was decreased in OPA lung tissues, as well as in cells overexpressing the envelope glycoprotein of JSRV (JSRV Env). Reduced numbers of autophagosomes were also observed in cells overexpressing JSRV Env, although assessment of autophagic flux showed that JSRV Env overexpression did not block the formation of autophagosomes, suggesting increased degradation of autolysosomes. Last, mouse xenograft experiments indicated that inhibition of autophagy by 3-methyladenine suppressed both tumor growth and the epithelial-to-mesenchymal transition. In conclusion, JSRV, through JSRV Env, takes advantage of the autophagy process, leading to the development of OPA.

Pathological Spectrum of Ovine Pulmonary Adenocarcinoma in Small Ruminants: A Focus on the Mixed Form.

Ovine pulmonary adenocarcinoma (OPA) is a contagious respiratory tumor of small ruminants, manifesting in chronic weight loss and respiratory failure. Infection with the betaretrovirus jaagsiekte sheep retrovirus (JSRV) is the cause of OPA. Here, we describe the gross and microscopic features of twenty-six sheep and one goat with naturally occurring JSRV-associated OPA. All the animals included in this study had pulmonary lesions morphologically consistent with OPA, but the majority of the observed lesions demonstrated features of both the classical and the atypical form of OPA, and were, therefore, classified grossly as mixed. The gross lesions were located mainly in the cranial pulmonary lobes, were multifocal to coalescing, variable in number and size, flat to slightly raised, firm, and white to grey. Histologically, the cases were classified according to the predominant architectural patterns as lepidic, papillary, acinar, or mixed; the mixed histological pattern was the most prevalent. The aim of this study was to describe the gross and microscopic spectrum of OPA in naturally infected small ruminants from Spain. The mixed form of OPA is less commonly reported, and can be confused with other concurrent pulmonary pathologies (such as BALT hyperplasia in SRLV-associated pneumonia or lungworm granulomas).

A Combined Approach for Detection of Ovine Small Ruminant Retrovirus Co-Infections.

Jaagsiekte retrovirus (JSRV)-induced ovine pulmonary adenocarcinoma (OPA) is an important ovine respiratory disease in Switzerland. Furthermore, ovine lungs with OPA frequently exhibited lesions suggestive of maedi-visna virus (MVV) or caprine arthritis encephalitis virus (CAEV) infection, indicating that co-morbidities might occur. Lungs and pulmonary lymph nodes were sampled from suspected OPA cases, inflammatory lung lesions and control lungs (total of 110 cases). Tissues were (a) processed for histology and immunohistochemistry (IHC), and (b) underwent DNA extraction and real-time PCR for JSRV, MVV and CAEV. Peptide sequences were used to generate virus-specific customized polyclonal antibodies. PCR-positive OPA cases and formalin-fixed and paraffin-embedded MVV- and CAEV-infected synovial cell pellets served as positive controls. Fifty-two lungs were histologically diagnosed with OPA. Histological evidence of MVV/CAEV infection was detected in 25 lungs. JSRV was detected by PCR in 84% of the suspected OPA cases; six were co-infected with MVV and one with CAEV. MVV was detected by PCR in 14 cases, and four lungs were positive for CAEV. Three lungs had MVV/CAEV co-infection. In IHC, JSRV was detected in 91% of the PCR-positive cases, whereas MVV and CAEV immunoreactivity was seen in all PCR-positive lungs. Although PCR showed a higher sensitivity compared to IHC, the combined approach allows for investigations on viral cell tropism and pathogenic processes in co-morbidities, including their potential interdependency. Furthermore, an immunohistochemical tool for specific differentiation of MVV and/or CAEV infection was implemented.

Endogenous Retroviruses Drive Resistance and Promotion of Exogenous Retroviral Homologs.

Endogenous retroviruses (ERVs) serve as markers of ancient viral infections and provide invaluable insight into host and viral evolution. ERVs have been exapted to assist in performing basic biological functions, including placentation, immune modulation, and oncogenesis. A subset of ERVs share high nucleotide similarity to circulating horizontally transmitted exogenous retrovirus (XRV) progenitors. In these cases, ERV-XRV interactions have been documented and include (a) recombination to result in ERV-XRV chimeras, (b) ERV induction of immune self-tolerance to XRV antigens, (c) ERV antigen interference with XRV receptor binding, and (d) interactions resulting in both enhancement and restriction of XRV infections. Whereas the mechanisms governing recombination and immune self-tolerance have been partially determined, enhancement and restriction of XRV infection are virus specific and only partially understood. This review summarizes interactions between six unique ERV-XRV pairs, highlighting important ERV biological functions and potential evolutionary histories in vertebrate hosts.

JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3' end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

[JSRV (jaagsiekte sheep retrovirus) and ovine pulmonary carcinoma]. / JSRV (jaagsiekte sheep retrovirus) et cancer du poumon associé chez le mouton.

JSRV (jaagsiekte sheep retrovirus) is a betaretrovirus, infecting small ruminants. This virus is responsible for the development of pulmonary adenocarcinoma, by the transformation of epithelial cells of the bronchioli and alveoli. This animal cancer is related to human bronchioloalveolar cancer (BAC), a specific form of human lung cancer for which a viral etiology has been proposed for several decades. In small ruminants JSRV interacts with the cells through the Hyal2 receptor. JSRV genome is simple and does not contain already known oncogene. It is now well established that the envelope protein is oncogenic by itself, via the cytoplasmic domain of the transmembrane glycoprotein and some domains of the surface glycoprotein. Activation of the PI3K/Akt and MAPK pathways participates to the envelope-induced transformation. The tumour development is associated with telomerase activation.

Lack of association between polymorphic copies of endogenous Jaagsiekte sheep retrovirus (enJSRVs) and Ovine Pulmonary Adenocarcinoma.

Ovine Pulmonary Adenocarcinoma (OPA) is a retrovirus-induced lung tumor of sheep, goat and mouflon, and its etiologic agent, Jaagsiekte sheep retrovirus (JSRV) is the only virus known to cause a naturally occurred lung adenocarcinoma. The oncogenic JSRV has several endogenous counterparts termed enJSRVs, some of which have been shown to interfere with JSRV replication at early and late stages of the retroviral cycle inhibiting JSRV exit from the cell, and thus, protecting sheep against the infection. In this work, Latxa sheep breed animals were classified depending on the presence/absence of OPA-characteristic clinical lesions in the lung. Using a PCR genotyping method and a logistic regression-based association study, five polymorphic enJSRV copies were analyzed in 49 OPA positive sheep and 124 control individuals. Our results showed that the frequency of the provirus enJSRV-16 is much higher in Latxa sheep breed than in other breeds, suggesting a recent proliferation of this provirus in the studied breed. However, no polymorphic enJSRV was found to be statistically associated with the susceptibility/resistance to OPA development.

Advances in the study of transmissible respiratory tumours in small ruminants.

Sheep and goats are widely infected by oncogenic retroviruses, namely Jaagsiekte Sheep RetroVirus (JSRV) and Enzootic Nasal Tumour Virus (ENTV). Under field conditions, these viruses induce transformation of differentiated epithelial cells in the lungs for Jaagsiekte Sheep RetroVirus or the nasal cavities for Enzootic Nasal Tumour Virus. As in other vertebrates, a family of endogenous retroviruses named endogenous Jaagsiekte Sheep RetroVirus (enJSRV) and closely related to exogenous Jaagsiekte Sheep RetroVirus is present in domestic and wild small ruminants. Interestingly, Jaagsiekte Sheep RetroVirus and Enzootic Nasal Tumour Virus are able to promote cell transformation, leading to cancer through their envelope glycoproteins. In vitro, it has been demonstrated that the envelope is able to deregulate some of the important signaling pathways that control cell proliferation. The role of the retroviral envelope in cell transformation has attracted considerable attention in the past years, but it appears to be highly dependent of the nature and origin of the cells used. Aside from its health impact in animals, it has been reported for many years that the Jaagsiekte Sheep RetroVirus-induced lung cancer is analogous to a rare, peculiar form of lung adenocarcinoma in humans, namely lepidic pulmonary adenocarcinoma. The implication of a retrovirus related to Jaagsiekte Sheep RetroVirus is still controversial and under investigation, but the identification of an infectious agent associated with the development of lepidic pulmonary adenocarcinomas might help us to understand cancer development. This review explores the mechanisms of induction of respiratory cancers in small ruminants and the possible link between retrovirus and lepidic pulmonary adenocarcinomas in humans.

Ovine pulmonary adenocarcinoma: a large animal model for human lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. Recent progress in understanding the molecular pathogenesis of this disease has resulted in novel therapeutic strategies targeting specific groups of patients. Further studies are required to provide additional advances in diagnosis and treatment. Animal models are valuable tools for studying oncogenesis in lung cancer, particularly during the early stages of disease where tissues are rarely available from human cases. Mice have traditionally been used for studying lung cancer in vivo, and a variety of spontaneous and transgenic models are available. However, it is recognized that other species may also be informative for studies of cancer. Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer of sheep caused by retrovirus infection and has several features in common with adenocarcinoma of humans, including a similar histological appearance and activation of common cell signaling pathways. Additionally, the size and organization of human lungs are much closer to those of sheep lungs than to those of mice, which facilitates experimental approaches in sheep that are not available in mice. Thus OPA presents opportunities for studying lung tumor development that can complement conventional murine models. Here we describe the potential applications of OPA as a model for human lung adenocarcinoma with an emphasis on the various in vivo and in vitro experimental systems available.

IFITMs restrict the replication of multiple pathogenic viruses.

The interferon-inducible transmembrane protein (IFITM) family inhibits a growing number of pathogenic viruses, among them influenza A virus, dengue virus, hepatitis C virus, and Ebola virus. This review covers recent developments in our understanding of the IFITM's molecular determinants, potential mechanisms of action, and impact on pathogenesis.

Jaagsiekte sheep retrovirus biology and oncogenesis.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology.