Clinical, molecular, and pathological investigations of ovine pulmonary adenocarcinoma in the middle of Iraq.

Background: Ovine pulmonary adenocarcinoma (OPA), caused by Jaagsiekte sheep retrovirus (JSRV), is a contagious neoplastic disease in sheep characterized by chronic respiratory signs, inducing the transformation of secretory epithelial cells of the distal respiratory tract. Aims: To perform clinical, epidemiological, and molecular studies with evaluation of some predisposing factors at the herd level of OPA infection in sheep in Al-Qadisiyah Province, Iraq. Methods: The first step of the study was undertaken to evaluate the clinical cases of OPA in naturally infected sheep and correlation with observing respiratory signs. Seventy-five sheep with chronic respiratory signs were examined clinically, and by molecular and sequences analysis. The second step was the epidemiological part that was carried out on 195 randomly selected animals from 30 flocks, with the prevalence rate based on PCR; sex, age, and size of flocks were assessed, as well as macroscopic and microscopic features of the neoplastic lung. Deep nasal swabs and nasal secretion were collected from all animals. RNA extraction and RT-PCR were also carried out. Results: The results showed that 12 (16%) samples were positive for OPA, based on env gene-specific primers. Nucleotide sequences of partial 545 bp of the env gene showed (0.07-0.12) variations from global strains presented in the NCBI database. The prevalence rate of OPA was 21/195 (10.76%) with PCR. The epidemiological factors analysis showed that there was no effect of sex and herd size on the prevalence rates (p ≥ 0.01), whereas age was significantly affected and the age of 2-4 years was more susceptible (p ≥ 0.01). Gross and microscopic examinations were discussed with the confirmation of an OPA infection. Conclusion: The current study provides useful data about the clinical and epidemiological features of JSRV that is circulating in sheep of Iraq, and concludes that epidemiological studies and disease control may require multi-diagnostic assays.

A survey of jaagsiekte sheep retrovirus (JSRV) infection in sheep in the three northeastern provinces of China.

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a "LHMKYXXM" motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the "LHMKYXXM" motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the "LHMKYXXM" motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.

Determination of reference genes for ovine pulmonary adenocarcinoma infected lung tissues using RNA-seq transcriptome profiling.

Ovine pulmonary adenocarcinoma (OPA) is a globally occurring tumor of lung epithelium which seriously affects the development of sheep farming. In our research, lung tissues of 3 naturally infected OPA individuals and 3 healthy individuals (2-4 years old) were collected. RNA was extracted for transcriptome analysis and reference gene selection. According to transcriptome analysis, 7 candidate reference genes (eukaryotic translation initiation factor 1, EIF1; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; beta-actin, ACTB; GABA Type A receptor-associated protein, GABARAP; activating transcription factor 4, ATF4; ribosomal protein S15, RPS15; and Y-Box binding protein 1, YBX1) showed fragments per kilobase of transcript per million fragments mapped (FPKM) values > 200.0 and standard errors of the means (SEM) < 20.0. Expression of the above candidate reference genes was evaluated by Real-time quantitative polymerase chain reaction (RT-qPCR) combined with the analysis using GeNorm, NormFinder, and BestKeeper software. Comprehensive analysis of the results showed that ACTB was the most stable one, followed by EIF1 and GABARAP. Then, expression stability of the above three genes were validated, suggesting as suitable reference genes in sheep lung tissue, in additional 30 OPA-affected lung tissues and 10 healthy ovine lung tissues. Finally, our findings will be helpful for the subsequent study on the tumorigenic mechanism of OPA.

Nasal adenocarcinoma associated with jaagsiekte sheep retrovirus infection in a sheep.

Betaretrovirus-induced transmissible respiratory tumors in sheep arise at 2 distinct anatomic locations, either deep in the lung tissue caused by jaagsiekte sheep retrovirus (JSRV) or in the nasal cavity induced by ovine enzootic nasal tumor virus (ENTV-1). JSRV and ENTV-1 are found in many countries worldwide and have a significant economic and animal health impact. Although JSRV is endemic in sheep in the British Isles, ENTV-1 has not been reported. We report herein a nasal adenocarcinoma in a cull 8-y-old Belclare ewe from Ireland. The gross and microscopic features and immunohistochemistry results were consistent with an ENTV-1-associated tumor. However, differential PCR, using primers specific to regions of divergent sequence between the viruses, was performed on different parts of the adenocarcinoma and produced consistent results: positive for JSRV and negative for ENTV-1. An association of JSRV with nasal adenocarcinoma in sheep has not been reported previously, to our knowledge. Our case shows the necessity of using PCR in combination with immunohistochemistry to reach an accurate etiologic diagnosis, which is of importance in countries currently free of ENTV-1.

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.

Expression of p53 protein, Jaagsiekte sheep retrovirus matrix protein, and surfactant protein in the lungs of sheep with pulmonary adenomatosis.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring cancer in sheep that is caused by the Jaagsiekte sheep retrovirus (JSRV). Because the pathologic and epidemiologic features of OPA are similar to those of bronchoalveolar carcinoma in humans, OPA is considered a useful animal model for pulmonary carcinogenesis. In this study, 3,512 lungs from various breeds of sheep were collected and macroscopically examined. OPA was identified in 30 sheep, and samples of these animals were further examined by histologic, immunohistochemical (p53 protein, surfactant protein A [SP-A], proliferating cell nuclear antigen [PCNA], JSRV matrix protein [MA]), and PCR methods. Papillary or acinar adenocarcinomas were detected microscopically in the affected areas. Immunoreactivity for p53 PAb240 was detected in 13 sheep, whereas p53 DO-1 was not detected in any of the OPA animals. PCNA immunoreactivity was recorded in 27 animals. SP-A and JSRV MA protein was immunopositive in all 30. JSRV proviral DNA was detected by PCR analysis in all of the lung samples collected from OPA animals. In addition, the pulmonary SP-A levels were increased in tumor cells. The results of this study suggest that PCNA and p53 protein expression may be useful indicators in monitoring malignancy of pulmonary tumors.

[Pathological Diagnoses and Whole-genome Sequence Analyses of the Jaagsiekte Sheep Retrovirus in Xinjiang, China].

To carry out pathologic diagnoses and whole-genome sequence analyses of the Jaagsiekte sheep retrovirus (JSRV) in Xinjiang, China, we first observed sheep suspected to have the JSRV. Then, the extracted virus suspension was observed by transmission electron microscopy (TEM). Total RNAs from lungs of JSRV-infected sheep were extracted and reverse-transcribed using a cDNA synthesis kit. Six pairs of primers were designed according to the exogenous reference virus strain (AF105220). Reverse transcription-polymerase chain reaction was carried out from JSRV-infected tissue, and the whole genome of the JSRV sequenced. Our results showed: flow of nasal fluid ("wheelbarrow test"); different sizes of adenoma lesions in the lungs; papillary hyperplasia of alveolar epithelial cells; alveolar cavity filled with macrophages; dissolute nuclei in central lesions. TEM revealed JSRV particles with a diameter of 88 nm to 125. 4 nm. The full-length of the viral genome sequence was 7456 bp. BLAST analyses showed nucleotide homology of 96% and 95% compared with that of the representative strain from the USA (AF105220) and UK (AF357971). Nucleotide homology was 89.8% and 89.9% compared with the endogenous Jaagsiekte sheep retrovirus, Inner Mongolia strain (DQ838493) and USA strain (EF680300). The specific pathogenic amino-acid sequence "YXXM" was found in the TM district, similar to the exogenous JSRV: this gene has been reported to be oncogenic. This is the first report of the complete genomic sequence of the exogenous JSRV from Xinjiang, and could lay the foundation for study of the biological characteristics and pathogenic mechanisms of the pulmonary adenomatosis virus in sheep.

Ovine pulmonary adenocarcinoma in slaughtered sheep: a pathological and polymerase chain reaction study.

Ovine pulmonary adenocarcinoma (OPA) is a contagious tumour in sheep caused by jaagsiekte sheep retrovirus (JSRV). This tumour originates from the pneumocyte type II and Clara cells and grossly appears as hard, prominent nodules in different lobes. The clinical signs of the disease are similar to those of other chronic respiratory diseases and are not pathogonomic. Therefore, post mortem examinations and histopathological studies are the most reliable ways to diagnose OPA, particularly subclinical cases of this neoplasm. In this study, out of 1000 sheep lungs grossly inspected, 50 animals were suspected of OPA. The suspected lungs as well as 25 apparently normal lungs were examined by histopathological and PCR methods. The proviral DNA was detected in 1/25 apparently normal lungs and 8/50 of the suspected lungs and subsequently confirmed by histopathological studies. The PCR-positive lung samples from five sheep revealed lesions of 'atypical' OPA and those from three sheep showed the 'classic' form of the disease. The tumours were multifocal and the masses were distributed throughout the cranioventral and diaphragmatic lung lobes. The stroma of the tumours in the atypical cases was more severely affected with inflammatory cell infiltration and connective tissue proliferation. The histopathological characteristics of maedi including hyperplasia of the perivascular and peribronchiolar lymphoid cells, interstitial lymphoplasmacytic infiltration and smooth muscle hyperplasia were also associated with OPA, especially the atypical form of this adenocarcinoma. Atypical OPA was more prevalent than the classic form. Geographic and climatic conditions, duration of exposure to the virus and the immune status of individual animals might be responsible for the differences between the two pathological entities of OPA.

Solitary tumours associated with Jaagsiekte retrovirus in sheep are heterogeneous and contain cells expressing markers identifying progenitor cells in lung repair.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). This study examines immunohistochemically solitary lung nodules considered as early OPA lesions from 11 sheep infected naturally by JSRV. All 11 neoplastic nodules exhibited features of adenocarcinoma and in four of them mesenchymal growth was also observed. Both types of lesion were labelled with antibody specific for JSRV-Env. In two cases infiltrating lymphoreticular cells also contained JSRV-Env. All tumours had a high Ki67 labelling index and variably contained cells expressing CC10 (a marker of Clara cells (CCs)), SPC (a marker of type II pneumocytes), p63 and keratin 14 (markers for stem/progenitor cells of the lung airway epithelia). Tumours with mesenchymal growth had intense expression of vimentin and desmin, weak expression of smooth muscle actin and did not express pancytokeratin and p63. Both epithelial and mesenchymal proliferations did not express the stem cell markers CD90 and CD117, but some tumour infiltrating cells expressed CD133. Solitary OPA tumours can therefore be adenocarcinomas or mixed tumours and have a heterogeneous cellular composition, containing groups of cells expressing markers that characterize local progenitor cells involved in lung repair.

Host species barriers to Jaagsiekte sheep retrovirus replication and carcinogenesis.

Understanding the factors governing host species barriers to virus transmission has added significantly to our appreciation of virus pathogenesis. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep that has rarely been found in goats. In this study, in order to further clarify the pathogenesis of OPA, we investigated whether goats are resistant to JSRV replication and carcinogenesis. We found that JSRV induces lung tumors in goats with macroscopic and histopathological features that dramatically differ from those in sheep. However, the origins of the tumor cells in the two species are identical. Interestingly, in experimentally infected lambs and goat kids, we revealed major differences in the number of virus-infected cells at early stages of infection. These differences were not related to the number of available target cells for virus infection and cell transformation or the presence of a host-specific immune response toward JSRV. Indeed, we also found that goats possess transcriptionally active endogenous retroviruses (enJSRVs) that likely influence the host immune response toward the exogenous JSRV. Overall, these results suggest that goat cells, or at least those cells targeted for viral carcinogenesis, are not permissive to virus replication but can be transformed by JSRV.

Long-term study of ovine pulmonary adenocarcinogenesis in sheep with marginal vs. sufficient nutritional selenium supply: results from computed tomography, pathology, immunohistochemistry, JSRV-PCR and lung biochemistry.

The impact of selenium (Se) in carcinogenesis is still debatable due to inconsistent results of observational studies, recent suspicion of diabetic side effects and e.g. dual roles of glutathione peroxidases (GPx). Previously, our group introduced long-term studies on lung carcinogenesis using the jaagtsiekte sheep retrovirus (JSRV) induced ovine pulmonary adenocarcinoma (OPA) as an innovative animal model. The present report describes the results of sufficient (0.2 mg Se/kg dry weight (dw)) vs. marginal (<0.05 mg Se/kg dw) nutritional Se supply on cancer progression over a two-year period in 16 animals. Computed tomography (CT) evaluation of lung cancer progression, final pathological examination, evidence of pro-viral JSRV-DNA in lung, lymph nodes and broncho-alveolar lavage cells as well as biochemical analysis of Se, GPx1 and thioredoxin reductase (TrxR) activity in lung tissue were recorded. Additionally, immunohistochemical determination of GPx1 expression in unaffected and neoplastic lung cells was implemented. The feeding regime caused significant differences in Se concentration and GPx1 activity in lung tissue between groups, whereas TrxR activity remained unaffected. JSRV was evident in broncho-alveolar lavage cells, lung tissue and lung lymph nodes. Quarterly executed CT could not demonstrate differences in lung cancer proliferation intensity. Necropsy and histopathology substantiated CT findings. Immunohistochemical analysis of GPx1 in lung tissue suggested a coherency of GPx1 immunolabelling intensity in dependence of tumour size. It was concluded that the model proved to be suitable for long-term studies of lung cancer proliferation including the impact of modifiable nutritional factors. Proliferation of OPA was unaffected by marginal vs. sufficient nutritional Se supply.

Pathological and aetiological studies in sheep exhibiting extrathoracic metastasis of ovine pulmonary adenocarcinoma (Jaagsiekte).

Seven sheep with a histopathological diagnosis of pulmonary adenocarcinoma with extrathoracic metastases were included in this retrospective study aiming to describe the pathological findings and to establish their relationship with Jaagsiekte sheep retrovirus (JSRV). In order of frequency, extrathoracic metastases were found in the liver, kidneys, skeletal muscle, digestive tract, spleen, skin and adrenal glands. Intrathoracic metastases involved the chest wall, regional lymph nodes, diaphragm and heart. Immunohistochemistry and polymerase chain reaction allowed detection of JSRV-related protein and nucleic acid, respectively, in the extrathoracic tumours of all cases. It is concluded that extrathoracic metastases constitute a pathological event of ovine pulmonary adenocarcinoma and confirm the malignant character of this virus-induced neoplasia.

Copy number variation and differential expression of a protective endogenous retrovirus in sheep.

The Jaagsiekte sheep retrovirus exJSRV and its endogenous counterpart enJSRV co-exist in sheep. exJSRV, a betaretrovirus, is the etiological agent of ovine pulmonary adenocarcinoma, and it has been demonstrated in vitro that an enJSRV Gag variant bearing the R-to-W amino acid change at position 21 was able to block exJSRV budding from the cells, providing a potential protective role for the host. In this work, we developed a fast mutation detection assay based on the oligo ligation assay (OLA) that permits the quantification of the relative proportions of the R21 and W21 Gag variants present in individual genomes and in cDNA obtained from normal and exJSRV-induced lung tumors. We have shown that the W21/R21 ratio is variable within and between breeds. We also describe for the first time that putative protecting enJSRV variants were expressed in alveolar type II cells (AECII), the major target of exJSRV.

Analysis of jaagsiekte sheep retrovirus (JSRV) envelope protein domains in transformation.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer in sheep. A unique feature is that JSRV envelope protein is also the oncogene for this virus. Previous studies have identified the cytoplasmic tail (CT) of the envelope transmembrane (TM) protein as critical for transformation although other regions of Env have also been implicated. In this study, the roles of other Env regions in transformation were investigated. Chimeras between JSRV Env and the Env of a related non-oncogenic endogenous retrovirus (enJSRV, 5F16) were used. A chimera containing the membrane-spanning region (MSR) of enJSRV inserted into JSRV Env showed substantially reduced transformation, indicating that the MSR plays a role in transformation. Transformation by this chimera was highly dependent on both Ras/Raf/MEK/MAPK and PI3K/Akt/mTOR signaling. A chimera containing the two amino acids in the TM ectodomain that distinguish JSRV and enJSRV showed modestly reduced transformation. Chimeras in the SU protein indicated that the amino terminal region of SU contributes to transformation, while the C-terminal part is not important. To test if Env trimerization is important for transformation, we mutated a leucine-rich sequence in the putative trimerization domain in the ectodomain of TM (Tri-M). This mutant could not transform cells and it did not oligomerize. However, Tri-M could complement a non-transforming mutant CT mutant (Y590F) so oligomerization is not necessary for at least some aspects of transformation. These experiments provide new insight into the regions and residues of JSRV Env protein necessary for oncogenic transformation.

An investigation of growth factors and lactoferrin in naturally occurring ovine pulmonary adenomatosis.

Ovine pulmonary adenomatosis (OPA), also known as jaagsiekte, is a transmissible beta retrovirus-induced lung tumour of sheep that has several features resembling human bronchoalveolar carcinoma (BAC). Angiogenesis has been suggested to be one of the most important factors underlying tumour growth and invasion. This process involves the action of growth factors including vascular endothelial growth factor (VEGF)-C, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-C and its receptor (PDGFR-α). Bovine lactoferrin (bLF), an iron and heparin-binding glycoprotein secreted into various biological fluids, has been implicated in innate immunity and has anti-inflammatory and anti-tumour functions. Tissues from 16 cases of OPA were compared with tissues from seven healthy control sheep by immunohistochemistry. Expression of the markers was assessed semi-quantitatively by ascribing an immunoreactivity score (IRS) with a maximum value of 300. VEGF-C, bFGF, PDGF-C, PDGFR-α and bLF signals were detected in 10/16, 15/16, 12/16, 15/16 and 10/16 of the OPA cases studied, respectively. bLF expression was weak in the neoplastic epithelial cells (IRS 21.4 ± 10.0) in contrast to high levels detected in infiltrating macrophages and plasma cells (IRS 141.3 ± 24.8 and 140.0 ± 25.1, respectively). The PDGFR-α IRS was elevated for neoplastic epithelial cells (108.9 ± 18.2) and was lowest for macrophages and plasma cells (20.4 ± 13.1 and 13.7 ± 12.4, respectively). These results suggest that bFGF, VEGF-C and PDGF-C have roles in the pathogenesis of OPA. bLF may activate macrophages and plasma cells in these lesions, but limited expression of bLF by neoplastic cells may be a consequence of defective or impaired function of this molecule.

Immunohistochemical and histopathological findings of ovine pulmonary adenocarcinoma (Jaagsiekte) in Egyptian sheep.

Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring retrovirus-induced transmissible lung cancer in sheep. Lungs and associated (bronchial and mediastinal) lymph nodes of seven sheep with OPA were examined. Lungs had few multifocal consolidated slightly elevated gray to white masses ranging from 0.5 to 3 cm in diameter. Histopathologically, these masses appeared as well-differentiated acinar adenocarcinoma with little evidence of anaplasia. The acini composed of well-differentiated cuboidal to low columnar epithelium with clear or vacuolated cytoplasm and low mitotic index. No metastases were observed in the bronchial and mediastinal lymph nodes of any animal. The presence of Jaagsiekte sheep retrovirus (JSRV) was demonstrated in the lungs by immunohistochemistry. JSRV protein was detected in all tumor epithelial cells, histologically normal alveolar type II cells, and few bronchiolar epithelial cells, alveolar macrophages, lymphocytes, and plasma cells. This study is the first to confirm the presence of natural OPA in Egypt.

Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair.

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer.

Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.

Ovine pulmonary adenocarcinoma as an animal model of progressive lung cancer and the impact of nutritional selenium supply.

Jaagsiekte sheep retrovirus (JSRV) is known to induce ovine pulmonary adenocarcinoma (OPA). Several studies have suggested an influence of selenium (Se) status on cancer progression. Thus, combining OPA with a defined Se supply might serve as a suitable animal model to study the impact of Se on lung cancer progression. 16 naturally JSRV-infected sheep were divided into 2 treatment groups receiving (a) <0.05 and (b) 0.2 mg Se/kg dry matter in diet, respectively. Computed tomography (CT) was performed repeatedly and evaluated using a CT-OPA-score system. Liver biopsies were taken three-monthly, blood samples were collected biweekly to study treatment effects on Se concentrations and glutathione peroxidase (GPx) activity. Cell pellets from bronchoalveolar lavage fluid (BALF) were tested for JSRV by PCR to approve the infection. To date, four animals of the ongoing study have been euthanised. Autopsy and histopathology were performed and correlated to CT analysis. JSRV was detected in BALF cell pellets. Progression of lung tumours was monitored successfully by repeated CT examinations, enabling the detection of even small nodules or increased lung density. Histopathology revealed bronchioloalveolar adenocarcinoma in lung areas suspicious to be OPA from CT evaluation. Score-based analysis of CT images for quantifying tumour progression proved as a valuable tool. Se concentration and GPx activity increased in liver and serum of group b and verified the efficiency of different feeding regime. In conclusion, OPA along with CT, autopsy/histopathology, trace element and enzyme activity analysis provide a suitable large animal model to examine the impact of Se supply on lung tumourigenesis.