To facilitate the commercialization of protein therapeutics, the establishment of a mass production system is necessary for cost reduction and ensuring a stable supply. The common method for producing protein therapeutics is a mammalian cell-based expression system, predominantly utilizing Chinese hamster ovarian (CHO) cells. In the process of generating stable CHO cell lines expressing protein therapeutics, the most critical step is the selection of high producer cell lines. While limiting dilution has been a classical method for selecting candidate clones, it is time-consuming and lacks the ability to evaluate cell productivity. To address this issue, in this study, a novel selection method utilizing Cell Picking System was established and compared with limiting dilution in identifying high-producer clones. In this method, transfected CHO cells were loaded into a microchamber containing 20-µm-diameter microwells, allowing secreted proteins from individual cells to be detected using fluorescently labeled antibodies. The results of small-scale production test demonstrated that clones with higher productivity distributions were enriched using Cell Picking System. In addition, the highest clone achieved 2.34 g/L protein production, suggesting potential applicability for commercial supply with some modifications in cell culture conditions. This method supports high-throughput applications of protein therapeutics to the industrial scale and addresses the limitations of traditional selection methods.

Glanders is a contagious zoonotic disease caused by Burkholderia mallei that primarily affects horses, donkeys, and mules. It remains endemic in parts of Asia, Africa, the Middle East, and South America, where increasing case reports highlight the need for accessible diagnostic tools. Conventional diagnostic methods are limited by high costs and specialized laboratory requirements, whereas lateral flow assays (LFAs) offer practical, user-friendly, and rapid alternatives. In this study, we developed three LFAs targeting antibodies against the B. mallei antigens Hcp1, GroEL, and whole-cell lysate (WCL), previously identified as promising markers in indirect enzyme-linked immunosorbent assays. Recombinant streptococcal protein G served as the colloidal gold conjugate. Diagnostic performance was evaluated using glanders-positive (n = 31) and glanders-negative (n = 51) equine sera collected in Mongolia. Hcp1-LFA demonstrated 100% sensitivity and 100% specificity, while WCL-LFA achieved 100% sensitivity and 98.0% specificity. GroEL-LFA showed 100% sensitivity but lower specificity (90.2%). Hcp1- and WCL-LFA also exhibited high sensitivity in asymptomatic horses. No cross-reactivity was observed with sera from horses with other infections (n = 60) collected in Japan. All LFAs remained functional for at least 16 weeks at 37°C. These results indicate that Hcp1- and WCL-based LFAs provide accurate, rapid, and equipment-free detection of glanders. Their simplicity, stability, and affordability make them highly suitable for field diagnosis, especially in low-resource endemic settings. Nonetheless, further validation using larger and more diverse sample sets is required to confirm their broader applicability. IMPORTANCE:Glanders is a contagious disease affecting horses, donkeys, and mules that can have serious economic and animal health consequences. Current diagnostic tests are expensive, time-consuming, and require specialized equipment. The assays developed in this study are simple, rapid, and cost-effective, allowing veterinarians to quickly identify infected animals in the field. Early detection helps prevent the spread of the disease, safeguarding livestock, supporting animal health, and reducing economic losses in affected regions.

Immune checkpoint inhibitors (ICIs) have emerged as a promising therapeutic strategy for canine oral malignant melanoma (OMM). However, only a subset of cases have shown clinical responses. Additionally, predictive biomarkers for the efficacy of ICIs in veterinary medicine are yet to be established. Tumour-infiltrating lymphocytes (TILs) are predictive biomarkers for ICI therapy in humans. In this study, we aimed to investigate the predictive utility of TILs for canine OMM treated with ICI therapy. Immunohistochemistry was performed on pre-treatment pathological tissues to detect cells positive for CD3 (pan-T cell marker), Granzyme B (cytotoxic T lymphocyte or natural killer cell marker) and Foxp3 (regulatory T cell marker). Further, we analysed the infiltration patterns of these cells and their prognostic utility for anti-programmed cell death ligand 1 (PD-L1) antibody therapy. There were variances in the infiltration levels of each TIL subset; further, we observed several TIL infiltration patterns in canine OMM. Survival analysis revealed that high infiltration of CD3+ and Granzyme B+ cells was significantly associated with prolonged progression-free survival and overall survival. Furthermore, the immune cell composition ratio, which reflected the balance between cytotoxic and regulatory cells, was significantly associated with survival time. These findings suggest that canine OMM exhibits immunological phenotypes analogous to those observed in human cancers. Taken together, the TIL profile holds significant potential as a prognostic biomarker for canine OMM treated with anti-PD-L1 antibody therapy.

Vaccination is highly effective in controlling infectious diseases in calves. A prime-boost injection is often administered intramuscularly in the neck and the hip in calves. However, no guidelines have established whether the same vaccination sites could be used for booster vaccinations. This study investigated the influence of intramuscular administration sites for sequential vaccines on immune responses in calves. All calves received the primary live-attenuated viral vaccine into the left side of the neck. Four weeks later, the booster was administered at the same site as the primary vaccine (local boost group) or in the right hip (distal boost group). The neutralizing antibody titers did not differ significantly between the groups. To assess T-cell responses to viral antigens after booster vaccination, the expression levels of lymphocyte activation markers CD25 and CD69 were measured in peripheral blood mononuclear cells (PBMCs). The site of booster administration had no significant effect on the activation of CD4+ and CD8+ T cells. Furthermore, vaccine-induced production of interferon-γ, tumor necrosis factor-α, and interleukin-6 in PBMC culture supernatants did not differ significantly between the groups. This study detected no significant differences in vaccine-specific immune responses between the local and distal booster vaccination sites in calves.

In enzootic bovine leukosis (EBL), a B-cell lymphoma caused by bovine leukemia virus (BLV) infection, immune remodeling within tumor-affected lymph nodes is currently poorly understood. Here, we analyzed chemokine production and macrophage phenotypes in tumor-affected lymph nodes in EBL, focusing on CCL4. Intracellular cytokine staining revealed that, compared with healthy lymph nodes, EBL tumor-affected lymph nodes showed increased proportions of CCL4 expressing cells, particularly among CD4+ T cells and B cells. Functional migration assays showed that recombinant CCL4 induced robust monocyte migration, with a comparatively modest T-cell migration. To identify lymph node macrophages, we characterized CD11bhiCD172a+ cells and confirmed their macrophage identity based on high expression levels of CD11c, CD14, CD16, and CD68. Using this definition, we found that the CD11bhiCD172a+ population was markedly increased in tumor-affected lymph nodes. Phenotypic analysis revealed an increased proportion of CD163+ and CD163+PD-L1+ macrophages, consistent with the acquisition of an immunosuppressive phenotype. In parallel, macrophages in tumor-affected lymph nodes showed a shift from MHC class I/IIhi to MHC I/IIlo subsets and reduced co-expression of MHC molecules with CD80 and CD86, indicating impaired antigen-presenting features. Collectively, these findings suggest that elevated CCL4 production in tumor lesions induced by EBL is associated with monocyte recruitment and the accumulation of phenotypically altered macrophages, highlighting a chemokine-driven remodeling of the tumor immune microenvironment.

The cattle tick Rhipicephalus microplus faces periods of nutritional stress that are inherent to its life cycle and during which starvation is a critical physiological challenge demanding specific metabolic adaptations. We used the embryonic cell line BME26 as a model to study metabolic responses during nutritional stress, to investigate the mechanisms underlying tick resilience. Our findings demonstrate that BME26 cells withstand prolonged starvation by activating gluconeogenesis, as evidenced by a marked increase in phosphoenolpyruvate carboxykinase (PEPCK) activity. Follow-up qPCR and enzymatic assays of tick tissues and BME26 cells identified the cytosolic PEPCK-C isoform as the predominantly, if not exclusively, expressed and active form, despite initial RNAseq data indicating transcripts for both PEPCK-C and PEPCK-M. Consistent with the predominant expression of the cytosolic form in R. microplus, the mitochondrial PEPCK-M isoform was present at minimal levels, exhibiting expression approximately 1,000 times lower than that of PEPCK-C. PEPCK silencing significantly reduced cell viability, particularly under starvation, demonstrating its critical role in energy homeostasis. The knockdown also downregulated the expression of fructose-1,6-bisphosphatase 1 (FBPase1) and glucose-6-phosphate dehydrogenase (G6PDH) while upregulating the autophagy-related gene ATG8. Additionally, the PEPCK inhibitor 3-mercaptopicolinic acid (3-MPA) at high concentrations caused decreased BME26 viability and reduced tick oviposition. Altogether, these results identify gluconeogenesis as a fundamental pathway for tick survival and a promising metabolic target for novel tick control strategies.

Ticks transmit diverse pathogens and secrete saliva with immunomodulatory properties that facilitates blood feeding. However, the cellular mechanisms underlying saliva-mediated immune suppression remain poorly defined. Here, we identify macrophages as the central hub through which saliva from the cattle tick Rhipicephalus microplus suppresses T-cell mediated immunity. Tick saliva inhibited IFN-γ and TNF production by CD4⁺ and CD8⁺ T cells while enhancing IL-10 and TGF-β expression, particularly in regulatory T cells. These effects were lost upon removal of CD14⁺ cells, underscoring the importance of macrophage-lineage cells in saliva-induced immunosuppression. Transcriptomic and functional analyses showed that tick saliva reprograms macrophages into an immunosuppressive state with reduced MHC class II, attenuated proinflammatory signaling, and suppressed chemokine production. Consistent with these findings, immunofluorescence analysis of tick-feeding sites showed an increased presence of IL-10-expressing macrophages and T cells. Our findings highlight a strategy by which arthropod saliva suppresses host immunity to facilitate pathogen transmission.

Horses develop spontaneous tumors, typically in old age. Although local tumor control can be achieved using conventional therapies, systemic therapies are required to treat recurrent and/or metastatic tumors. Immune checkpoint inhibitors, such as anti-PD-L1 antibodies, have been approved for the treatment of various tumor types in humans; however, little is known about the immunosuppressive roles of the PD-1/PD-L1 pathway in horses, and the therapeutic potential of these inhibitors remains to be elucidated. Previously, we reported that the rat monoclonal anti-PD-L1 antibody 6C11-3A11 cross-reacts with horse PD-L1 to block the PD-1/PD-L1 interaction. To further develop antibodies for therapeutic purposes, their immunogenicity must be reduced to maximize efficacy and safety. To this end, we designed an equinized (equine-ized) anti-PD-L1 antibody, Eq6C11, using the complementarity-determining regions of 6C11-3A11. Eq6C11 had antigen-binding properties comparable to those of 6C11-3A11 and inhibited equine PD-L1 binding to PD-1 in a recombinant protein-based assay. Treatment with Eq6C11 significantly increased IFN-γ and IL-2 production in equine peripheral blood mononuclear cell cultures, suggesting its stimulatory activity on T-cell activation. Although further studies are needed to clarify its immunogenicity and clinical activity, these results encourage further development of Eq6C11 as a candidate immune checkpoint inhibitor for cancer immunotherapy in horses.

The cattle tick Rhipicephalus microplus is an ectoparasite of major economic importance to livestock, particularly in tropical and subtropical regions. The lack of effective vaccines against tick infestation has hindered its control and the identification of new molecular targets for recombinant vaccine development arises as an alternative strategy. Tick saliva is known as a reservoir of potential vaccine targets, including several proteins involved in attachment, immune response modulation and feeding that targeting might affect tick infestations. Among these proteins, histamine binding proteins (HBPs) sequester host histamine molecules reducing itching and pain response, allowing that ticks attach to the host and get a complete blood meal. In this study, we aimed to functionally and immunologically characterize a putative HBP from R. microplus (BrRm-HBP) and evaluate its potential as an anti-tick vaccine. In silico analyses indicated that BrRm-HBP is conserved among tick species and presents structural features consistent with known tick HBPs. Immunohistochemical assays demonstrated its localization in salivary glands, supporting its potential role at the tick-host interface. Recombinant BrRm-HBP was expressed in Escherichia coli and showed histamine-binding activity. Immunization of rabbits with recombinant BrRm-HBP elicited a humoral immune response. Moreover, vaccination conferred partial protection in rabbits experimentally infested with the closely related tick Rhipicephalus linnaei. These findings indicate that BrRm-HBP is a functional and immunogenic salivary protein potentially involved in tick-host interactions and may represent a promising candidate for new anti-tick vaccine formulations.

Marek's disease virus (MDV) causes lymphomas and neurological disorders in chickens. Although vaccination largely controls outbreaks, highly virulent strains continue to emerge. The major MDV-encoded oncoprotein is Meq, functions as a transcription factor. Amino acid polymorphisms in Meq have been reported to influence virulence. Despite routine vaccination, MD still occurs sporadically in Japan. Japanese isolates harbour characteristic Meq polymorphisms, but their impact on MDV virulence remains unclear. We investigated the transcriptional regulation by Meq from Japanese isolates and evaluated the pathogenicity of recombinant MDV (rMDV) encoding Meq from the Japanese isolate Nr-c1. Nr-c1-Meq exhibited reduced transrepression and transactivation on viral gene promoters. An rMDV encoding Nr-c1-Meq (vNr-c1-Meq) induced lower mortality and tumourigenicity than an rMDV encoding Meq from the parental very virulent RB-1B strain (vRB-1B). vNr-c1-Meq did not cause visceral tumours or neurological disorders but resulted in distinct clinical signs, including open-mouth breathing. In lymphoid tissues from vNr-c1-Meq-infected chickens, a lower proportion of CD4+ T cells, the targets of MDV transformation, and lower viral loads were observed than those in vRB-1B-infected chickens. Histopathological examination revealed increased lymphocyte infiltration in bronchus-associated lymphoid tissues (BALT) in the vNr-c1-Meq group. Additionally, flow cytometric analysis showed elevated γδ T-cell proportions, which positively correlated with IFN-γ expression in vNr-c1-Meq-infected chickens and were linked to BALT hyperplasia. In comparison to the vRB-1B, vNr-c1-Meq infection resulted in attenuated disease progression and altered clinical signs. These findings suggest that Meq polymorphisms not only influence MD virulence but also clinical presentation.

The tumor suppressor protein p53 is extensively characterized as a regulator of genome integrity, apoptosis, and metabolic homeostasis in mammalian systems. In early-diverging metazoans, however, p53 metabolic functions remain poorly understood. Here, we investigated the metabolic responses of BME26 cells, an embryonic cell line derived from the cattle tick Rhipicephalus microplus, exposed to PRIMA-1. Pharmacological treatment using PRIMA-1 reduced BME26 cell viability in a dose- and time-dependent manner, exhibiting similar effects to those observed in human breast cancer cells, which were utilized as comparative reference models displaying distinct PRIMA-1 response profiles. Annexin V/7-AAD staining and morphological analyses showed that the decrease in BME26 viability was primarily caused by apoptosis. Metabolomic profiling by 1H-13C HSQC NMR revealed coordinated changes in central carbon metabolism, amino acid pathways, and choline-derived metabolites, indicating broad metabolic remodeling upon PRIMA-1 exposure. Despite showing no effect on glucose uptake and glycogen content, PRIMA-1 selectively reduced the expression of key glycolytic genes, including hexokinase (HK) and pyruvate kinase (PK), and significantly reduced glucose-6-phosphate dehydrogenase (G6PDH) gene expression. This was accompanied by decreased intracellular NADPH levels, while isocitrate dehydrogenase-1 (IDH-1) gene expression remained unaffected, suggesting a pathway-specific metabolic modulation rather than global energetic collapse. Collectively, our findings demonstrate that PRIMA-1 treatment in tick embryonic cells restricts glycolytic and pentose phosphate pathway flux, limiting NADPH production and reshaping redox balance to favor apoptosis. These results support the potential conservation of metabolic pathways sensitive to PRIMA-1, which in mammalian systems are linked to modulation of the p53-G6PDH-NADPH axis, thereby expanding our understanding of metabolic regulation and stress responses in arthropod biology.

Tick saliva contains bioactive molecules that can modulate host immune responses and facilitate blood feeding. Among these, prostaglandin E2 (PGE2) has been implicated in host immunosuppression, but its temporal dynamics during feeding and functional relevance remain unclear. In this study, we quantified PGE2 concentrations in Rhipicephalus microplus saliva across different feeding stages and assessed its immunomodulatory effects on bovine macrophages. Salivary PGE2 levels showed stage-dependent changes, with the highest concentrations detected during the mid-feeding phase. PGE2 suppressed TNF-α production from macrophages stimulated with LPS and IFN-γ in a concentration-dependent manner. Notably, saliva samples with higher endogenous PGE2 concentrations induced stronger suppression of TNF-α. Furthermore, the immunosuppressive effect of PGE2 was replicated by selective agonists against the two PGE2 receptors EP2 and EP4, suggesting a receptor-mediated mechanism. In addition, the saliva-mediated suppression of TNF-α production was significantly reversed by pharmacological blockade of EP receptors. Our findings reveal that the temporal increase in salivary PGE2 during tick feeding is functionally linked to suppression of host macrophage activation. These results suggest that PGE2 contributes to the immunomodulatory activity of tick saliva during blood feeding.

Marek’s disease virus (MDV) causes Marek’s disease (MD) in chickens, which is characterized by malignant lymphomas and neurological disorders. Although MD is currently controlled using live vaccines, the virulence of field strains has continuously increased in recent decades. Polymorphisms in the MDV-encoded oncoprotein Meq are shared among field strains according to their virulence. In particular, very virulent MDV strains harbor characteristic amino acid changes in the basic region of Meq at positions 77 and 80; however, the contribution of these polymorphisms to virulence remains unclear. To assess the impact of these polymorphisms on MDV virulence, we generated recombinant MDV (rMDV) based on the very virulent RB-1B strain, harboring K77E and D80Y substitutions in Meq found in low-virulent strains (rRB-1B_Meq77/80). Chickens were challenged with rMDVs, and survival rates and tumor incidence were evaluated. Viral loads in major organs were quantified by quantitative PCR, and the dynamics of MDV-infected cells and T cells were analyzed using flow cytometry. In addition, histopathological analysis was performed to further examine differences in pathogenesis in detail. To elucidate the mechanisms underlying pathogenesis, we conducted reporter assays to assess the effect of these polymorphisms in the basic region on its transcriptional regulatory activity. rRB-1B_Meq77/80 exhibited a reduced virulence but unexpectedly caused other clinical signs, including open-mouth breathing, in infected chickens. Quantitative PCR analysis showed consistently lower viral loads across all examined organs in rRB-1B_Meq77/80-infected chickens. Flow cytometric analysis revealed a reduction in MDV-infected cells, accompanied by a notable increase in CD8⁺ T cell populations. Histopathological analysis showed bronchus-associated lymphoid tissue hyperplasia in the lungs. Reporter assays revealed that most amino acid substitutions in the basic region in low-virulence strains reduced transcriptional regulatory activity. Our data indicate that polymorphisms at positions 77 and 80 in the Meq of low-virulence strains reduce MDV virulence and Meq-mediated transcription and possibly alter pathogenesis. This study improves our understanding of the mechanisms underlying MDV virulence.

Bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) are retroviruses infecting cattle, with BLV being a known cause of enzootic bovine leukosis. In contrast, BIV is considered non-pathogenic and has been less studied, despite its relevance to animal lentiviral evolution. This study aimed to develop and validate a multiplex real-time PCR assay for simultaneous detection of BIV proviral DNA and quantification of BLV proviral load. Novel primers and probes targeting the BIV pol gene were designed. The assay demonstrated a detection limit of 10 copies per reaction with a probability of 83%, comparable to conventional nested PCR. Importantly, the addition of BIV primers and probes did not interfere with the accurate quantification of BLV. Furthermore, BIV detection was unaffected even in samples with high BLV proviral loads, confirming the assay's robustness for simultaneous detection. The validated assay was applied to a molecular epidemiological survey of 6,051 cattle in the Kyushu region of Japan, conducted between 2021 and 2025. No BIV proviral DNA was detected in any sample, indicating a low prevalence of BIV in this population. This multiplex PCR assay provides a sensitive, efficient, and labor-saving tool for integrated surveillance of retroviral infections in cattle. Furthermore, it offers a valuable platform for future studies on the epidemiology and ecological role of BIV in bovine populations.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a B-cell lymphoma in cattle. Previous studies have demonstrated that T cells of BLV-infected cattle show increased expression of immune checkpoint molecules, including programmed death-1 (PD-1), lymphocyte-activation gene-3 (LAG-3), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and T-cell immunoglobulin domain and mucin domain-3 (TIM-3), leading to T-cell exhaustion. However, the key immune checkpoint molecules driving T-cell exhaustion in BLV-induced tumorigenesis remained unclear. In this study, we identified the key immune checkpoint molecules by performing comprehensive flow cytometric analyses of T cells from EBL cattle, and elucidated the phenotype and function of exhausted T cells using a transcriptomic analysis by RNA sequencing and cell culture assays. The comprehensive expression analysis revealed that the proportion of CD4+ and CD8+ T cells co-expressing PD-1 and TIM-3 was significantly increased in the peripheral blood and tumor tissues of EBL cattle compared to healthy cattle. Transcriptomic analysis of PD-1+TIM-3+ T cells revealed the upregulation of genes related to terminal exhaustion and the downregulation of genes related to T-cell differentiation and response in this subset. Additionally, PD-1+TIM-3+ T cells exhibited higher expression of CTLA-4, LAG-3, and Eomes, and lower expression of T-bet, suggesting a terminally exhausted phenotype. Cell culture assays revealed a significant impairment in IFN-γ production in PD-1+TIM-3+ T cells upon stimulation, reflecting severe dysfunction. These findings indicate that PD-1+TIM-3+ T cells play a central role in T-cell exhaustion during BLV-induced tumorigenesis. This study provides valuable insights for future therapeutic strategies against BLV infection.

Bovine leukemia virus (BLV) infects B cells in ruminants and causes lymphoma after an extended latency period. Previous studies have demonstrated T-cell exhaustion through the upregulation of immunoinhibitory molecules, including programmed death-ligand 1 (PD-L1) and T-cell immunoglobulin and mucin domain-3 (TIM-3), in BLV-infected cattle. However, studying T-cell exhaustion across all BLV infection stages remains challenging due to the virus’s prolonged latency in cattle. Sheep provide a valuable model, as they develop lymphoma more rapidly than cattle. This study examined PD-L1 and TIM-3 expression kinetics and T-cell function in BLV-infected sheep. During persistent infection, PD-L1 expression was correlated with BLV proviral load. TIM-3 expression increased in CD4+, CD8+, and γδTCR+ T cells. Functional analysis revealed that TIM-3 blockade enhanced T-cell activation markers (CD25 and CD69) in cultured PBMCs from infected sheep and increased CD69+IFN-γ+ and CD69+TNF-α+ populations, particularly among CD4+ T cells. Combined PD-L1 and TIM-3 blockade significantly enhanced cytokine production in both CD4+ and CD8+ T cells, while PD-L1 blockade alone showed limited effects. These findings demonstrate the effect of TIM-3 blockade in restoring immune function during chronic BLV infection, effective both alone and in combination. This study validates sheep as a valuable model for investigating immune checkpoint dynamics and evaluating immunotherapies for BLV infection and other chronic diseases.

A combination of irradiation and oclacitinib, a Janus kinase (JAK) inhibitor used in dogs, could lead to synergistic anticancer effects in canine tumors. However, the anti-tumor effects of oclacitinib remain unclear. This study investigated the radio-sensitizing effect of oclacitinib in canine tumors and determined its underlying mechanisms using osteosarcoma (HMPOS), malignant melanoma (CMeC), and thyroid adenocarcinoma (CTAC) cell lines. A clonogenic assay and a tumor growth assessment in a xenograft mouse model (BALB/cAJcl-nu/nu) were performed to evaluate the radio-sensitizing effects of oclacitinib. Oclacitinib enhanced the radio-sensitivity of tumor cells both in vitro and in vivo. The signal transducer and activator of transcription (STAT)3 expression was activated and suppressed by oclacitinib in X-irradiation exposed cells. Oclacitinib enhanced radiation-induced apoptosis only in HMPOS cells by inhibiting anti-apoptotic genes. In addition, oclacitinib inhibited the transcription of cell cycle-regulating genes and arrested cell cycle progression from the G1 phase to subsequent phases. In conclusion, oclacitinib enhanced radio-sensitivity both in vitro and in vivo by triggering apoptosis and impeding cell cycle progression via STAT3 inhibition in canine tumor cell lines. This study suggested the clinical therapeutic potential of oclacitinib and radiation therapy in enhancing treatment efficacy and outcomes in canine tumors.

Bovine leukemia virus (BLV), a retrovirus that is widespread worldwide, causes enzootic bovine leukosis (EBL), a B-cell leukemia/lymphoma with a poor prognosis that ultimately results in death. In Japan, the number of cattle infected with this virus is increasing, and it is estimated more than 35% of cattle are currently infected. Since no vaccines or treatments against BLV infection are currently available, it is important to establish a method of early diagnosis for EBL to reduce economic losses caused by the disposal of EBL cattle in Japan, where a large number of expensive beef cattle are raised. We previously developed Rapid Amplification of the Integration Site without Interference by Genomic DNA Contamination (RAISING), a cost-effective, rapid, and sensitive method for the clonality analysis of BLV-infected cells. Despite its usefulness for the early diagnosis of EBL, RAISING had drawbacks preventing its practical application. Here, we report the development of an improved method, RAISING ver.2, and its performance. Compared to BLV clonality analysis using the previous method, RAISING ver.2 was found to maintain high accuracy and reproducibility despite its simplification. Moreover, its performance was also validated in a multicenter validation study. Taken together, our results strongly suggest that RAISING ver.2 can be fully utilized in clinical practice. Successful commercialization of a RAISING test kit could overcome the concerns of livestock farmers suffering from EBL, thereby promoting a stable supply of Japanese beef, both domestically and internationally.

Marek’s disease virus (MDV) is the etiological agent of Marek’s disease (MD), a lymphoproliferative disorder in chickens. Polymorphisms in the MDV-encoded oncoprotein Meq are shared among field strains and correlate with their virulence. The attenuated vaccine strain CVI988 harbors unique amino acid polymorphisms in Meq, particularly at positions 71, 77, and 326. In this study, we investigated the impact of these polymorphisms on Meq protein function and MDV virulence. Reporter assays revealed that the substitutions, particularly A71S and K77E, markedly impaired the transcriptional regulatory activity of Meq. To evaluate their effect on virulence, we generated a recombinant MDV based on the very virulent RB-1B strain, encoding Meq with A71S and K77E substitutions (rRB-1B_Meq71/77). Chickens infected with rRB-1B_Meq71/77 developed neither clinical signs nor lymphomas. Flow cytometry revealed no expansion of infected cells in this group, but a marked increase in CD8+ T and γδ T cells during early infection. Histopathological analysis also confirmed the absence of MD-associated lesions. These findings demonstrate that the polymorphisms at positions 71 and 77 in the CVI988 strain are sufficient to abolish MDV virulence. This study provides insight into the molecular basis of MDV virulence and informs the strategy for the design of more effective vaccines.

Marek's disease virus (MDV) causes lymphomas (Marek's disease) in chickens. Despite vaccination, MDV field strains exhibit increased virulence, and sporadic outbreaks are still reported. An insertion/deletion in Meq has been identified in several MDV strains, and our previous study using recombinant MDV (rMDV) demonstrated that an insertion in Meq enhanced MDV virulence, whereas a deletion reduced its virulence. However, the mechanisms by which these indels in Meq alter MDV virulence remain elusive. We aimed to clarify the impact of the insertion in Meq on pathogenesis. We compared the effects of Meq and Meq with the insertion, termed L-Meq, on transcriptional regulation, the dynamics of transformed T cells and relevant T-cell subsets, and the patterns of gene expression in tumor lesions of infected chickens. Reporter assays revealed that insertion increased the transactivation activity on the infected-cell protein 4 promoter in the MDV genome and bcl-2/cd30 promoters in the host genome. rMDV encoding L-Meq (vRB-1B_L-Meq) exhibited higher mortality and tumor incidence than rMDV encoding Meq (vRB-1B_Meq), and vRB-1B_L-Meq infection increased the proportion of CD4+ T cells, which are targets for transformation by MDV, in the early post-infection phase, compared with vRB-1B_Meq. RNA-seq analysis revealed that similar genes were modulated in tumor lesions of chickens infected with vRB-1B_Meq or vRB-1B_L-Meq. These findings suggest that although the insertion in Meq does not alter the target genes for transcriptional regulation, it accelerates the process of tumorigenesis by enhancing the transactivation activity.IMPORTANCEMarek's disease, an avian lymphoproliferative disease, is caused by Marek's disease virus (MDV). Meq, an MDV oncoprotein, regulates the expression of viral and host genes. Meq with an insertion, termed L-Meq, has been identified as a factor that enhances MDV virulence. However, the mechanisms by which the insertion in Meq alters MDV virulence remain unknown. Our study clarified that the insertion enhances the transactivation activity of Meq on the host promoters related to tumorigenesis. Notably, the transcriptomes of tumor lesions in chickens infected with recombinant MDV (rMDV) with L-Meq and those infected with rMDV encoding Meq without the insertion were similar; however, chickens infected with rMDV harboring L-Meq exhibited higher proportions of CD4+ T cells and regulatory T cells, which are targets for transformation by MDV, in the early post-infection phase, suggesting accelerated tumorigenesis. This study contributes to the current understanding of the mechanisms underlying MDV virulence.