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.

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 Jaagsiekte sheep retrovirus envelope protein promotes sheep trophoblast cell fusion by activating PKA/MEK/ERK1/2 signaling.

BACKGROUND: Endogenous Jaagsiekte sheep retrovirus envelope protein (enJSRV-Env) plays an important role in trophoblast cell fusion in sheep. However, the underlying mechanism remains unclear. METHODS: Primary endometrial luminal epithelial cells (LECs) were isolated from the sheep uterus and cocultured with sheep trophoblast cells (STCs). Giemsa staining was conducted to count multinucleated cells in the coculture system. Gain- and loss-of-function assays were performed to explore the role of enJSRV-Env in trophoblast cell fusion in the coculture system. Co-immunoprecipitation and mass spectrometry were carried out to identify the interacting partner of enJSRV-Env in the cocultures. Western blot analysis were conducted to determine the activation of protein kinase A (PKA)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. RESULTS: Primary LECs were identified by the expression of epithelial marker cytokeratin 18. Overexpression of enJSRV-Env promoted the formation of multinucleated cells in the coculture system. enJSRV-Env activated and physically interacted with PKA, along with the activation of MEK/ERK1/2 signaling. PKA inhibition completely reversed enJSRV-Env-induced MEK/ERK1/2 activation, and ERK1/2 inhibition abolished enJSRV-Env-induced formation of multinucleated cells in the coculture system. CONCLUSION: enJSRV-Env promotes trophoblast cell fusion in the sheep placenta by activating PKA/MEK/ERK1/2 signaling. This finding reveals a novel mechanism underlying the contribution of enJSRV-Env to trophoblast cell fusion during placental morphogenesis.

A novel strategy for developing vaccine candidate against Jaagsiekte sheep retrovirus from the envelope and gag proteins: an in-silico approach.

BACKGROUND: Sheep pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by the Jaagsiekte retrovirus (JSRV). OPA typically has a serious economic impact worldwide. A vaccine has yet to be developed, even though the disease has been globally spread, along with its complications. This study aimed to construct an effective multi-epitopes vaccine against JSRV eliciting B and T lymphocytes using immunoinformatics tools. RESULTS: The designed vaccine was composed of 499 amino acids. Before the vaccine was computationally validated, all critical parameters were taken into consideration; including antigenicity, allergenicity, toxicity, and stability. The physiochemical properties of the vaccine displayed an isoelectric point of 9.88. According to the Instability Index (II), the vaccine was stable at 28.28. The vaccine scored 56.51 on the aliphatic index and -0.731 on the GRAVY, indicating that the vaccine was hydrophilic. The RaptorX server was used to predict the vaccine's tertiary structure, the GalaxyWEB server refined the structure, and the Ramachandran plot and the ProSA-web server validated the vaccine's tertiary structure. Protein-sol and the SOLPro servers showed the solubility of the vaccine. Moreover, the high mobile regions in the vaccine's structure were reduced and the vaccine's stability was improved by disulfide engineering. Also, the vaccine construct was docked with an ovine MHC-1 allele and showed efficient binding energy. Immune simulation remarkably showed high levels of immunoglobulins, T lymphocytes, and INF-γ secretions. The molecular dynamic simulation provided the stability of the constructed vaccine. Finally, the vaccine was back-transcribed into a DNA sequence and cloned into a pET-30a ( +) vector to affirm the potency of translation and microbial expression. CONCLUSION: A novel multi-epitopes vaccine construct against JSRV, was formed from B and T lymphocytes epitopes, and was produced with potential protection. This study might help in controlling and eradicating OPA.

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.

To scan or not to scan? Efficacy of transthoracic ultrasonography for ovine pulmonary adenocarcinoma screening in a large commercial UK sheep flock.

BACKGROUND: Transthoracic ultrasonography (TTUS) is currently the only widely used method to diagnose preclinical or subclinical ovine pulmonary adenocarcinoma (OPA) in the live sheep. However, little is known about the test characteristics of TTUS. METHODS: One thousand and seventy-four breeding ewes in a flock with evidence of low OPA prevalence underwent TTUS by an experienced operator. Fifty-one sheep were diagnosed with OPA and underwent gross postmortem examination (PME). RESULTS: Lesions consistent with OPA were found in only 24% (12/51) of the culled ewes. Thirty-five percent (18/51) of culled ewes had gross lesions consistent with other pulmonary disease and 41% (21/51) had no detectable gross lesions on PME. Histopathology and immunohistochemistry confirmed OPA in only the 12 animals identified with OPA lesions from PME. CONCLUSION: Great caution should be exercised when deciding if TTUS is an appropriate screening test in groups of sheep where OPA prevalence may be anticipated to be low. TTUS is a subjective test and thus individual operator ability will influence the sensitivity and specificity of TTUS for OPA diagnosis while the underlying prevalence influences the eventual positive predictive value.

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep.

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV-positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.

Insertion of an endogenous Jaagsiekte sheep retrovirus element into the BCO2 - gene abolishes its function and leads to yellow discoloration of adipose tissue in Norwegian Spælsau (Ovis aries).

BACKGROUND: The accumulation of carotenoids in adipose tissue leading to yellow fat is, in sheep, a heritable recessive trait that can be attributed to a nonsense mutation in the beta-carotene oxygenase 2 (BCO2) gene. However, not all sheep breeds suffering from yellow fat have this nonsense mutation, meaning that other functional mechanisms must exist. We investigated one such breed, the Norwegian spælsau. RESULTS: In spælsau we detected an aberration in BCO2 mRNA. Nanopore sequencing of genomic DNA revealed the insertion of a 7.9 kb endogenous Jaagsiekte Sheep Retrovirus (enJSRV) sequence in the first intron of the BCO2 gene. Close examination of its cDNA revealed that the BCO2 genes first exon was spliced together with enJSRV-sequence immediately downstream of a potential -AG splice acceptor site at enJSRV position 415. The hybrid protein product consists of 29 amino acids coded by the BCO2 exon 1, one amino acid coded by the junction sequence, followed by 28 amino acids arbitrary coded for by the enJSRV-sequence, before a translation stop codon is reached. CONCLUSIONS: Considering that the functional BCO2 protein consists of 575 amino acids, it is unlikely that the 58 amino acid BCO2/enJSRV hybrid protein can display any enzymatic function. The existence of this novel BCO2 allele represents an alternative functional mechanism accounting for BCO2 inactivation and is a perfect example of the potential benefits for searching for structural variants using long-read sequencing data.

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.

Exogenous Small Ruminant Betaretrovirus Envelope Protein Is Detected in Draining Lymph Nodes in Contagious Respiratory Tumors of Sheep and Goats.

Contagious respiratory tumors of sheep and goats are epithelial neoplasms of the lung and nasal cavities. They are associated with oncogenic betaretroviruses known as jaagsiekte sheep retrovirus and enzootic nasal tumor retrovirus of sheep and goats. We investigated the presence of the envelope protein (ENV) of these retroviruses in retropharyngeal and mediastinal lymph nodes using a specific monoclonal antibody by immunohistochemistry methods, single-labeled or combined with ovine B or T lymphocytes or macrophage cell markers. Samples of lymph nodes, fixed in formalin and zinc fixative, were obtained from paraffin-embedded material. Four groups of samples were used: 24 natural cases of ovine pulmonary adenocarcinoma (OPA), 13 of enzootic nasal adenocarcinoma of sheep (ENAS), 19 of enzootic nasal adenocarcinoma of goats (ENAG), and 14 control samples. ENV was detected by single labeling in cortical lymphoid follicles. Six of 24 OPA samples were positive and only in those from sheep with extensive neoplasia. Immunolabeling was detected in 5/13 ENAS and 10/19 ENAG samples. Positive labeling was found either in the intercellular spaces, membranes, or cytoplasm of cells in follicles. Control samples were not correspondingly labeled. Double immunohistochemistry demonstrated co-labeling of ENV and CD21 (B cells and follicular dendritic cells) in all samples, CD14 (macrophage) in OPA samples, and Pax-5 (B cells) in ENAG samples, but not with CD8 or CD4 (T lymphocytes). These results demonstrate the presence of betaretrovirus ENV proteins in nontumor cells in regional lymph nodes in sheep and goats with contagious respiratory tumors.

Enzootic nasal tumor virus type 2 envelope of goats acts as a retroviral oncogene in cell transformation.

Enzootic nasal tumor virus type 1 (ENTV-1) (ovine nasal tumor virus) and ENTV-2 (caprine nasal tumor virus) are known to be causative agents of enzootic nasal adenocarcinoma (ENA) in sheep and goats, respectively. Although the nucleotide and amino acid sequences of ENTV-1 and ENTV-2 are quite similar, they are recognized as phylogenetically distinct viruses. The envelope protein of ENTV-1 functions as an oncoprotein in the in vitro transformation of epithelial cells and fibroblasts. Thus, it is the primary determinant of in vivo tumorigenesis in ENA. As per our knowledge, no previous studies have reported in detail the role of ENTV-2 in ENA tumorigenesis. Here, in order to investigate the molecular mechanism of caprine ENA oncogenesis by ENTV-2, we have attempted to identify the transforming potential of ENTV-2 envelope, and investigated the activation of cell signaling pathways in oncogenic transformation. Our findings confirmed that ENTV-2 envelope was capable of inducing oncogenic transformation of rat cell lines in vitro. Further, we found that MAPK, Akt, and p38 were constitutively activated in ENTV-2 envelope-transformed clone cells. In addition, inhibitor experiments revealed that MEK-MAPK and PI3K-Akt signaling pathways are involved in the ENTV-2 envelope-induced cell transformation. These data indicate that ENTV-2 envelope could induce oncogenic transformation by signaling pathways that are also utilized by ENTV-1 envelope.

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.

Exogenous Jaagsiekte Sheep Retrovirus type 2 (exJSRV2) related to ovine pulmonary adenocarcinoma (OPA) in Romania: prevalence, anatomical forms, pathological description, immunophenotyping and virus identification.

BACKGROUND: Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by exogenous Jaagsiekte Sheep Retrovirus (exJSRV). The prevalence of JSRV-related OPA in Eastern European countries, including Romania is unknown. We aimed to investigate: the prevalence and morphological features of OPA (classical and atypical forms) in the Transylvania region (Romania), the immunophenotype of the pulmonary tumors and their relationships with exJSRV infection. A total of 2693 adult ewes slaughtered between 2017 and 2019 in two private slaughterhouses from Transylvania region (Romania) was evaluated. Lung tumors were subsequently assessed by cytology, histology, immunocytochemistry, immunohistochemistry, electron microscopy and DNA testing. RESULTS: Out of 2693 examined sheep, 34 had OPA (1.26% prevalence). The diaphragmatic lobes were the most affected. Grossly, the classical OPA was identified in 88.24% of investigated cases and the atypical OPA in 11.76% that included solitary myxomatous nodules. Histopathology results confirmed the presence of OPA in all suspected cases, which were classified into acinar and papillary types. Myxoid growths (MGs) were diagnosed in 6 classical OPA cases and in 2 cases of atypical form. Lung adenocarcinoma was positive for MCK and TTF-1, and MGs showed immunoreaction for Vimentin, Desmin and SMA; Ki67 expression of classical OPA was higher than atypical OPA and MGs. JSRV-MA was identified by IHC (94.11%) in both epithelial and mesenchymal cells of OPA. Immunocytochemistry and electron microscopy also confirmed the JSRV within the neoplastic cells. ExJSRV was identified by PCR in 97.05% of analyzed samples. Phylogenetic analysis revealed the presence of the exJSRV type 2 (MT809678.1) in Romanian sheep affected by lung cancer and showed a high similarity with the UK strain (AF105220.1). CONCLUSIONS: In this study, we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPA in sheep. Additionally, we described the first report of atypical OPA in Romania, and to the best of our knowledge, in Eastern Europe. Finally, we showed that MGs have a myofibroblastic origin.

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.

The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism.

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract-the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV-ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.

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.

Old origin of a protective endogenous retrovirus (enJSRV) in the Ovis genus.

Sheep, the Jaagsiekte sheep retrovirus (JSRV) and its endogenous forms (enJSRVs) are a good model to study long-time relationships between retroviruses and their hosts. Taking advantage of 76 whole genome resequencing data of wild and domestic Ovis, we investigated the evolution of this relationship. An innovative analysis of re-sequencing data allowed characterizing 462 enJSRVs insertion sites (including 435 newly described insertions) in the Ovis genus. We focused our study on endogenous copies inserted in the q13 locus of chromosome 6 (6q13). Those copies are known to confer resistance against exogenous JSRV thanks to alleles bearing a mutation in the gag gene. We characterized (i) the distribution of protective and non-protective alleles across Ovis species and (ii) the copy number variation of the 6q13 locus. Our results challenged the previous hypothesis of fixation and amplification of the protective copies in relation with domestication, and allowed building a new model for the evolution of the 6q13 locus. JSRV would have integrated the 6q13 locus after the Ovis-Capra divergence (5-11 MYA) and before the Ovis diversification (2.4-5 MYA). The protective mutation in the enJSRV 6q13 copy appeared shortly after its insertion and was followed by genomic amplifications, after the divergence between Pachyform lineage on one side and the Argaliform and moufloniform lineages on the other (2.4-5 MYA). Considering the potential selective advantage of the protective mutation, its fixation in both sheep and its closest wild relative Ovis orientalis may be due to natural selection before domestication from O. orientalis populations.

Blocked expression of key genes of the angiogenic pathway in JSRV-induced pulmonary adenocarcinomas.

JSRV (Jaagsiekte Sheep Retrovirus) is a retrovirus inducing a transmissible lung adenocarcinoma in sheep and goats with predominantly lepidic and papillary lesions. This naturally occurring lung cancer in large animals shares many features with human pneumonic-type lung adenocarcinomas with predominant lepidic growth. The metastatic spread is rare in both human and animal cancers. This unique feature prompted us to decipher the angiogenesis pathway in these cancers. We focused on the levels of mRNA and proteins of genes implicated in the extension of JSRV-induced lung adenocarcinomas by studying their expression in lung cancers (n = 10) and normal lungs (n = 10) and in primary epithelial alveolar type II cells derived from cancers (n = 10) or normal lungs (n = 6). In parallel, we evaluated the levels of expression of key genes in lung tissues collected from lepidic (n = 13) or papillary (n = 5) human adenocarcinomas and, when available, adjacent normal lungs (n = 11). We measured the expression of the same key genes implicated in angiogenesis, lymphangiogenesis and degradation of the extracellular matrix. In ovine adenocarcinomas, VEGFR2 and VEGFD mRNA were downregulated in cancers; MMP9, TIMP1 and FGFR2 mRNA were overexpressed as compared to normal lungs. Importantly, VEGFA and VEGFR2 proteins were not expressed in JSRV-induced cancers. In human lepidic adenocarcinomas, VEGFA and VEGFR2 mRNA were weakly expressed and no VEGFR2 protein was detectable. Downregulation of key angiogenic players may contribute to the control of extra thoracic invasion of cancer cells in human and ovine pneumonic-type adenocarcinoma with predominant lepidic growth.