Ovine pulmonary adenocarcinoma (OPA) is an infectious, neoplastic lung disease of sheep that causes significant animal welfare and economic issues throughout the world. Understanding OPA pathogenesis is key to developing tools to control its impact. Central to this need is the availability of model systems that can monitor and track events after Jaagsiekte sheep retrovirus (JSRV) infection. Here, we report the development of an experimentally induced OPA model intended for this purpose. Using three different viral dose groups (low, intermediate and high), localised OPA tumour development was induced by bronchoscopic JSRV instillation into the segmental bronchus of the right cardiac lung lobe. Pre-clinical OPA diagnosis and tumour progression were monitored by monthly computed tomography (CT) imaging and trans-thoracic ultrasound scanning. Post mortem examination and immunohistochemistry confirmed OPA development in 89% of the JSRV-instilled animals. All three viral doses produced a range of OPA lesion types, including microscopic disease and gross tumours; however, larger lesions were more frequently identified in the low and intermediate viral groups. Overall, 31% of JSRV-infected sheep developed localised advanced lesions. Of the sheep that developed localised advanced lesions, tumour volume doubling times (calculated using thoracic CT 3D reconstructions) were 14.8 ± 2.1 days. The ability of ultrasound to track tumour development was compared against CT; the results indicated a strong significant association between paired CT and ultrasound measurements at each time point (R2 = 0.799, p < 0.0001). We believe that the range of OPA lesion types induced by this model replicates aspects of naturally occurring disease and will improve OPA research by providing novel insights into JSRV infectivity and OPA disease progression.

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.

The jaagsiekte sheep retrovirus (JSRV), belonging to the betaretrovirus genus of the retroviridae family, includes both exogenous and endogenous jaagsiekte sheep retroviruses (exJSRV and enJSRV, respectively). At the proviral genome level, exJSRV and enJSRV strains have a high degree of similarity with their main variation regions being the LTR, gag, and env genes. In this study, for the first time, we investigated and compared the distribution of CpG islands between these enJSRV and exJSRV strains. Specifically, we analyzed a total of 42 full-length JSRV genomic sequences obtained from the GenBank® database to identify CpG islands in the exJSRV and enJSRV genomes using the MethPrimer software. Our results showed that the CpG islands in the two JSRV strains were mainly distributed in the LTR, gag, and env genes. In exJSRVs, 66.66% (6/9), 33.33% (3/9), and 100% (9/9) of the sequences presented at least one CpG island in LTR, gag, env genes, respectively, and for enJSRVs, 84.84% (28/33), 57.57% (19/33), and 96.96% (32/33) of the sequences presented at least one CpG island in the LTR, gag, and env genes. These findings suggested that the distribution, length, and genetic traits of CpG islands were different for the exJSRV and enJSRV strains. In future, it would be necessary to demonstrate the biological significance of CpG islands within these genes in exJSRV and enJSRV genomes. This will enhance understanding regarding the potential role of CpG islands in epigenetic regulation.

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.

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.
CONCLUSIONS: 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.

暂无摘要。

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.
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.
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.
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.
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.

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.
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).
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.
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 (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.

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.