BACKGROUND: Ovine pulmonary adenocarcinoma (OPA) is an infectious lung tumour caused by the Jaagsiekte Sheep Retrovirus. Histological examination is the cornerstone of OPA diagnosis and provides the final morphological basis for diagnosis. However, traditional pathology faces challenges, such as complex image interpretation and reliance on subjective judgment. Ensemble learning models have been increasingly applied to medical image classification. In this study, we constructed a dataset of 69,592 images (OPA: 33,609; non-OPA: 35,983) and divided it by employing a phased dataset division strategy. After evaluating DenseNet, EfficientNet, Res2Net101, and ResNet152, Res2Net101 was selected as the best-performing base model, and ensemble learning was conducted using two strategies: output-layer fusion (Efficient-Res2Net-L) and feature fusion (Efficient-Res2Net). Model performance was evaluated using accuracy, precision, Recall, and F1 score. Anti-peeking validation was conducted using five whole-slide images (three OPA, two non-OPA) not included in the dataset. An additional 600 image blocks were used to compare performance of the model with that of pathologists. RESULTS: Res2Net101 achieved the highest accuracy (94.3%) on the test set, whereas EfficientNet made the fewest misjudgements (11) in the anti-peeking image verification. EfficientNet also outperformed others in the comparison with pathologists (accuracy: 95.0%, specificity: 91.3%, sensitivity: 98.7%). The output-layer fusion model Efficient-Res2Net-L slightly outperformed feature fusion. Efficient-Res2Net showed improved accuracy (96.5%), specificity (93.7%), and sensitivity (99.3%), surpassing the performance of junior pathologists and approaching the performance of senior pathologists, with differences reduced to 2.3% and 5%, respectively. CONCLUSION: The integrated model Efficient-Res2Ne demonstrates high accuracy and robustness. Suspicious lesion areas can be identified through rapid initial diagnosis of tissue slice images, assisting pathologists in efficiently completing the final histological diagnosis. This is a valuable tool for improving diagnostic workflow efficiency.

Mitochondria are highly dynamic organelles involved in energy production, metabolic regulation, calcium homeostasis, apoptosis, and innate immunity. The mitochondrial network is susceptible to damage from physiological and environmental factors, including viral infections. Mitochondrial quality control (MQC) is the primary pathway that maintains normal physiological functions and mitochondrial homeostasis. Mitochondrial dynamics and mitophagy are complex processes within the MQC mechanism that can be exploited by viruses to modulate mitochondrial morphology, metabolism, and innate immune responses, achieving immune evasion, promoting self-replication, and accelerating infection. Viruses or their proteins target mitochondrial dynamics or mitophagy and regulate these processes via direct or indirect mechanisms. In addition, numerous molecular modulators of MQC have been reported. These findings provide new opportunities to understand the MQC process and have the potential for use as antiviral therapeutic agents. This article reviews the relationships between MQC, viral infection events, and viral pathogenesis, introduces the known molecular pharmacological regulators of MQC, and emphasizes their importance in antiviral drug development.

Ovine pulmonary adenocarcinoma (OPA), caused by the exogenous Jaagsiekte sheep retrovirus (JSRV), shares several pathological and molecular features with human lung adenocarcinoma, providing an important model for comparative oncology. JSRV pathogenesis is mostly studied at the transcriptome level, with systematic proteomic and metabolomic studies remaining insufficient. Therefore, this study aimed to systematically characterise the molecular alterations associated with JSRV-induced OPA by integrating direct data-independent acquisition proteomics and untargeted metabolomics. We established an OPA model by infecting lambs with JSRV and performed multi-omics analyses on the lesion and control lung tissues (n = 3 per group for both proteomic and metabolomic analyses). In total, 1631 differentially expressed proteins and 748 differential metabolites were identified, and the two omics datasets exhibited highly coordinated variations. Integrated analyses suggested that adhesion remodelling (with downregulated LIMCH1), endoplasmic reticulum stress (with upregulated HYOU1), and metabolic and immune-related alterations (accompanied by elevated GFPT1 and PTGS2) represent major biological processes associated with JSRV infection. Western blot analysis confirmed the expression changes in these proteins. Overall, this study provides a multidimensional molecular landscape of OPA and expands current understanding of the molecular alterations associated with JSRV infection. These findings provide candidate pathways and molecular targets for future mechanistic studies and comparative investigations of lung adenocarcinoma.

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma (OPA), and its pathogenesis is primarily mediated by the viral envelope (Env) protein. However, the detailed oncogenic mechanisms underlying JSRV infection remain incompletely understood. In this study, we integrated transcriptomic and metabolomic analyses to characterize JSRV Env-induced alterations in human bronchial epithelial BEAS-2B (cells). BEAS-2B cells transfected with the pcDNA4.0myc-his-JSRV-env plasmid, those transfected with the empty pcDNA4.0myc-his vector, and untreated BEAS-2B cells served as the experimental, negative control, and blank control groups, respectively. Transcriptomic analysis identified a total of 2733 differentially expressed genes (DEGs). Specifically, relative to the blank and negative control groups, 1178 genes were upregulated and 307 were downregulated in the JSRV-env group. These DEGs were significantly enriched in pathways related to altered cellular energy metabolism, cell cycle regulation, and oncogenic signaling. Metabolomic analysis revealed 451 differentially expressed metabolites (DEMs), with 192 detected in positive ion mode and 259 in negative ion mode. Compared with both control groups, the JSRV-env group exhibited 33 upregulated and 46 downregulated DEMs in positive ion mode, as well as 22 upregulated and 55 downregulated DEMs in negative ion mode. These DEMs were significantly enriched in pathways such as cellular metabolism, purine metabolism, amino acid metabolism, and the tricarboxylic acid cycle. Notably, cellular and mitochondrial energy metabolism pathways were closely linked. Analyses of mitochondrial- and mitophagy-related genes, alongside an integrated transcriptomic and metabolomic evaluation of their interactions, suggested mitochondrial damage and the potential activation of mitophagy. Furthermore, JSRV Env-transformed BEAS-2B cells exhibited elevated reactive oxygen species, decreased mitochondrial membrane potential, and abnormal mitochondrial morphology—characterized by swelling, as well as fragmented, dissolved, or disappearing cristae—along with the presence of myelin-like mitochondrial lesions and mitophagosomes. Mitochondrial and lysosomal probe co-localization further confirmed mitochondrial degradation in the transformed cells. Overall, these results highlight the potential involvement of altered mitochondrial energy metabolism and mitophagy in JSRV Env-induced BEAS-2B cell transformation. These findings offer novel insights into the mechanisms of viral oncoproteins, cellular metabolic reprogramming, and mitophagy, while identifying potential targets for understanding JSRV pathogenesis.

Introduction:Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumor caused by the exogenous Jaagsiekte sheep retrovirus (exJSRV). Analysing the genome of the pathogen is crucial for developing OPA prevention and control measures. Due to the absence of exogenous genomic JSRV-related information in Inner Mongolia, we aimed to establish a specific technique for exJSRV genomic amplification. Methods:Target virions were purified using U3 hn-PCR (hemi-nested PCR) combined with density gradient centrifugation. Specific reverse transcription primers were designed using the low-identity region of the internal and external genome, combined with long fragment PCR and 3'RACE technology, and the full-length genome of exogenous JSRV from Inner Mongolia was successfully obtained. Results:Exogenous molecular characteristics were found in the long terminal repeat(LTR)-U3 region, gag-variable region 1/2(VR1/VR2) and env-VR3, and was 98.8% identical to the Chinese JSRV-C1, which was significantly higher than that of foreign isolates (93.05-95.84%) and enogenous Jaagsiekte sheep retrovirus (enJSRV) (88.73-92.26%). Phylogenetic analysis showed that NMJS12 and exogenous JSRV-C1 were located in the same evolutionary clade. Accordingly, the whole genome eukaryotic expression plasmid was successfully constructed and viral particle packaging was achieved in 293T cells. Conclusion:Altogether, this study represents the first elucidation of the complete genome of exogenous JSRV in Inner Mongolia, China and provides a critical material foundation for antiviral target screening and research on OPA pathogenesis.

Rabies continues to pose a significant global zoonotic threat. In recent years, the increased spillover events of rabies viruses from wildlife to domestic animals have raised public health security concerns, prompting heightened international attention toward rabies management in wildlife populations. Our study reveals the first documented case of a rabies virus (RABV) strain isolated from Eurasian badgers (Meles meles) within Chinese ecosystems. Genetic analysis shows 99.4% nucleotide identity with dominant bovine-associated cosmopolitan lineages, offering robust evidence of interspecies transmission from wildlife reservoirs to domestic livestock. It is noteworthy that due to the special geographical location of this region, the habitat of Eurasian badgers overlaps with the territory of livestock and human settlements, thereby forming a transmission chain of rabies virus such as "fox- Eurasian badger-livestock" or "Eurasian badger-livestock." This critical finding highlights an urgent need for enhanced pathogen surveillance programs in pastoral regions where intensive human-wildlife-livestock interfaces create high-risk transmission zones.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour caused by the Jaagsiekte Sheep Retrovirus (JSRV). Histopathological diagnosis is the gold standard for OPA diagnosis. However, interpretation of traditional pathology images is complex and operator dependent. The mask regional convolutional neural network (Mask R-CNN) has emerged as a valuable tool in pathological diagnosis. This study utilized 54 typical OPA whole slide images (WSI) to extract 7167 typical lesion images containing OPA to construct a Common Objects in Context (COCO) dataset for OPA pathological images. The dataset was categorized into training and test sets (8:2 ratio) for model training and validation. Mean average specificity (mASp) and average sensitivity (ASe) were used to evaluate model performance. Six WSI-level pathological images (three OPA and three non-OPA images), not included in the dataset, were used for anti-peeking model validation. A random selection of 500 images, not included in the dataset establishment, was used to compare the performance of the model with assessment by pathologists. Accuracy, sensitivity, specificity, and concordance rate were evaluated. The model achieved a mASp of 0.573 and an ASe of 0.745, demonstrating effective lesion detection and alignment with expert annotation. In Anti-Peeking verification, the model showed good performance in locating OPA lesions and distinguished OPA from non-OPA pathological images. In the random 500-image diagnosis, the model achieved 92.8% accuracy, 100% sensitivity, and 88% specificity. The agreement rates between junior and senior pathologists were 100% and 96.5%, respectively. In conclusion, the Mask R-CNN-based OPA diagnostic model developed for OPA facilitates rapid and accurate diagnosis in practical applications.

Abstract Background Maedi-visna virus (MVV) is a lentivirus that infects monocyte/macrophage lineage cells in sheep, goats, and wild ruminants and causes pneumonia, mastitis, arthritis, and encephalitis. The immune response to MVV infection is complex, and a complete understanding of its infection and pathogenesis is lacking. This study investigated the in vivo transcriptomic patterns of lung tissues in sheep exposed to MVV using the RNA sequencing technology. Result The results indicated that 2,739 genes were significantly differentially expressed, with 1,643 downregulated genes and 1,096 upregulated genes. Many variables that could be unique to MVV infections were discovered. Gene Ontology analysis revealed that a significant proportion of genes was enriched in terms directly related to the immune system and biological responses to viral infections. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the most enriched pathways were related to virus-host cell interactions and inflammatory responses. Numerous immune-related genes, including those encoding several cytokines and interferon regulatory factors, were identified in the protein-protein interaction network of differentially expressed genes (DEGs). The expression of DEGs was evaluated using real-time polymerase chain reaction and western blot analysis. CXCL13, CXCL6, CXCL11, CCR1, CXCL8, CXCL9, CXCL10, TNFSF8, TNFRSF8, IL7R, IFN-γ, CCL2, and MMP9 were upregulated. Immunohistochemical analysis was performed to identify the types of immune cells that infiltrated MVV-infected tissues. B cells, CD4+ and CD8+ T cells, and macrophages were the most prevalent immune cells correlated with MVV infection in the lungs. Conclusion Overall, the findings of this study provide a comprehensive understanding of the in vivo host response to MVV infection and offer new perspectives on the gene regulatory networks that underlie pathogenesis in natural hosts.

IMPORTANCE:Ovine pulmonary adenomatosis (OPA) and maedi-visna disease (MVD) are chronic and progressive infectious diseases in sheep caused by Jaagsiekte sheep retrovirus (JSRV) and maedi-visna virus (MVV), respectively. OBJECTIVE:To investigate the pathological changes and conduct viral gene analysis of OPA and MVD co-occurrence in Inner Mongolia, China. METHODS:Using gross pathology, histopathology, immunohistochemistry, ultrastructural pathology, PCR, and sequence analysis, we investigated the concurrent infection of JSRV and MVV in 319 Dorper rams slaughtered in a private slaughterhouse in Inner Mongolia, in 2022. RESULTS:Of the 319 rams included, 3 showed concurrent JSRV and MVV infection. Gross lung pathology showed diffuse enlargement, consolidation, and greyish-white miliary nodules on the lung surface; the trachea was filled with a white foamy fluid; hilar and mediastinal lymph nodes were significantly enlarged. Histopathology results revealed typical OPA and MVD lesions in the lung tissue. Immunohistochemical results were positive for JSRV envelope protein (Env) in the tumor cells and MVV CA in alveolar macrophages. Transmission electron microscopy showed several virions and autophagosomes in the lung tissue, severely damaged mitochondria, and the induced mitophagy. Nucleotide sequences obtained for JSRV env and MVV gag showed the highest homology with the Inner Mongolian strains of JSRV env (JQ837489) and MVV gag (MW248464). CONCLUSIONS AND RELEVANCE:Our study confirmed that OPA and MVD co-occurrence and identified the pathological changes in Inner Mongolia, China, thereby providing references for the identification of concurrent JSRV and MVV infections.

To construct a lentiviral vector overexpressing JSRV-env,screen human bronchial epithe-lial cells-2b(BEAS-2B)stably expressing Env,and detect the effect of Env on cell proliferation and migration.The target gene env was amplified by PCR and inserted into the pMT194 plasmid to construct the pMT194-JSRV-env lentiviral overexpression plasmid.The recombinant plasmid was mixed with the packaging auxiliary plasmid and co-transfected with the transfection reagent com-plex into 293T cells.After collection,purification and concentration of the recombinant lentivirus,the virus was used to infect BEAS-2B cells,the infection conditions were optimized.The stable cell strain was screened by puromycin(Puro)according to the resistance gene puro carried by the lentiviral vector.The expression of the target gene was detected by qPCR and Western blot at the transcriptional and translational levels.The cell proliferation ability was detected by CCK-8 and the cell migration ability was detected by scratch test.The target gene env was successfully amplified and inserted into the PMT194 vector.The enzyme digestion identification was consistent with the expectation,and the PMT194-JSRV-env expression vector was successfully constructed.The ex-pression of env in 293T cells was significantly higher than that in blank control group and negative control group(P＜0.001).The average titer of JSRV Env recombinant lentivirus was 1.03 × 109 IU/mL.The optimalmultiplicity of infection(MOI)of the recombinant lentivirus on BEAS-2B cells was 40,the optimal Puro concentration for screening cell strain was 0.3 mg/L,and fluores-cence microscopy showed that BEAS-2B cells expressed red fluorescence.The results of qPCR and Western blot showed that the expression of env in the experimental group was significantly higher than that in the blank control group and the negative control group(P＜0.001).The Env-FLAG fu-sion protein was successfully expressed in the host cells.Compared with the blank control group and the negative control group,the proliferation and migration ability of the cells in the experimen-tal group were significantly enhanced(P＜0.05).In summary,the JSRV-env lentiviral overexpres-sion vector was successfully constructed,the BEAS-2B cell strain stably expressing Env was screened,and Env can significantly increase the proliferation and migration of BEAS-2B cells.

为实现对梅迪-维斯纳病毒(MVV)的快速检测,根据MVV gag基因保守序列设计特异性引物和探针,通过优化反应条件和反应体系,建立了 MVV的实时荧光RPA检测方法,并通过灵敏度、特异性、重复性试验及样品复核检测进行评价.结果显示:该方法最佳反应温度为39℃,用时短,20 min即可完成检测;灵敏度高,检测下限可达10-1 pg/μL,比常规PCR检测结果灵敏约10 倍;特异性强,与小反刍兽疫病毒、羊败血性链球菌、牛肠道病毒、传染性鼻气管炎病毒、牛副流感病毒3型、肺炎支原体等继发呼吸系统症状病原无交叉反应;重复性好,对于相同质量浓度的模板DNA检测变异系数均小于10％;可有效检测出MVV,与PCR结果一致.综上所述,本研究建立的MVV 荧光RPA检测方法适用于MVV的临床快速检测,可为该病的诊断及防控提供依据.

[目的]制备绵羊梅迪-维斯纳病毒(maedi-visna virus,MVV)衣壳蛋白(capsid protein,CA)多克隆抗体,并鉴定其特异性.[方法]根据MVV内蒙古分离株CA基因序列设计特异性引物,扩增CA基因,构建重组质粒;对CA重组蛋白进行原核表达及纯化,制备兔源MVV CA重组蛋白多克隆抗体,采用间接ELISA方法测定其抗体效价,利用Western blot和免疫组化方法对其进行特异性鉴定.[结果]成功构建MVV CA重组蛋白原核表达系统,纯化后的目的蛋白大小约27 kDa;间接ELISA方法测定制备的多克隆抗体效价为1:8192;Western blot检测感染MVV绵羊的病肺组织,在25 kDa处出现特异性条带;免疫组化结果显示感染MVV绵羊的病肺中巨噬细胞的胞浆内有明显棕黄色阳性信号.[结论]利用获得的可溶性重组MVV CA蛋白制备的多克隆抗体具有较好特异性,可为MVV血清学诊断技术提供检测抗体.

本研究旨在构建绵羊肺腺瘤病毒(JSRV)的囊膜基因(env)原核表达载体,制备多克隆抗体并对其特异性进行鉴定.以笔者所在实验室构建的pEGFP-C1/JSRV-env质粒为模板,PCR扩增env基因全长1 848 bp,构建原核表达质粒并命名为pET-32a/JSRV-env,进行双酶切鉴定和测序分析后,转化到E.coli Transetta(DE3),经IPTG诱导及亲和层析法对其纯化.将纯化蛋白常规免疫成年新西兰大白兔制备多克隆抗体,通过Western-blot方法鉴定多克隆抗体的特异性,免疫组化方法评价其临床检测效果.双酶切鉴定及测序结果表明,已成功构建pET-32a/JSRV-env重组质粒且包含编码YXXM基序的基因序列,成功表达及纯化该蛋白,其大小约89ku.以制备的多克隆抗体为一抗,经Western-blot检测自然感染绵羊肺腺瘤病(OPA)的病肺组织,在89 ku处出现单一特异性条带.免疫组化结果显示,OPA病肺肿瘤细胞可见棕黄色的阳性信号.结果表明,成功构建JSRV-env原核表达质粒,制备的多克隆抗体特异性较好,可用于生产实践中OPA的检测,且制备简便、成本低廉.同时也为进一步探讨JSRV Env的功能提供了实验基础.

Background Ovine pulmonary adenomatosis (OPA) is a contagious lung epithelial tumor of sheep caused by jaagsiekte sheep retrovirus (JSRV), which causes severe economic losses for the sheep industry in the world. The specific oncogenic mechanism of JSRV is not yet clarified. Methods In this study, RNA was extracted from lung tissues of 3 naturally infected OPA cases and 3 healthy individuals for transcriptome sequencing (RNA-Seq). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to confirm the sequencing data. Immunohistochemistry (IHC) and western blot (WB) were performed to confirm the signaling pathway enriched by DEGs that was activated in naturally infected OPA cases. Cell viability, wound-healing, transwell and colony formation assays were performed to assess the cell malignant transformation of sheep trophoblast cells (STCs) transformed with JSRV- env lentivirus in vitro, and then WB was performed to confirm the signaling pathway that had been validated in the lung tissues. Results A total of 366 DEGs (154 up-regulated and 212 down-regulated) were identified by RNA-Seq of lung tissues of naturally infected OPA cases and healthy individuals. GO analysis showed that 366 DEGs were significantly enriched in 178 GO terms, including 114 biological processes, 19 cellular components and 45 molecular functions. KEGG analysis showed that the DEGs mainly enriched in cell proliferation, differentiation, apoptosis and migration, such as PI3K/Akt/mTOR, MAPK and Hippo signaling pathway, and Hippo signaling pathway has never been reported in naturally infected OPA cases. qRT-PCR results of 10 DEGs which were selected randomly were consistent with RNA-Seq results. The protein expression of Hippo signaling pathway were up-regulated in naturally infected OPA lung tissues. Cell viability, wound-healing, transwell and colony formation assays confirmed that JSRV- env lentivirus caused malignant transformation of STCs and JSRV Env increased the protein expression of Hippo signaling pathway. Conclusions This research first identified the changes in the transcriptome level of naturally infected OPA lung tissues. These data confirm that the Hippo signaling pathway is involved in the mechanism of OPA, clarify the interaction between Hippo signaling pathway and JSRV Env, provide further evidence for the tumorigenic mechanism of JSRV.