Search Results (2,543)

Kobuviruses infect a wide range of hosts and exhibit high genetic diversity, highlighting the need for further studies to better understand their evolution and impact on animal health. In the present study, bovine kobuvirus (BKoV) was detected in 40 of 154 fecal samples from calves using the primers targeting the 3D gene region, corresponding to an overall positivity rate of 25.97%. All positive samples were obtained from diarrheic calves younger 30 days of age, (40/133; 30.08%), consistent with previous studies. To enable more reliable genotyping, the complete VP1 gene sequences of seven BKoV strains were obtained, and the phylogenetic analyses showed that all VP1 sequences of this study clustered within Aichivirus B1. Additionally, immunoinformatics analysis of the VP1 protein identified conserved linear and discontinuous B-cell epitopes that may represent potential targets for cross-reactive diagnostic assays or future vaccine studies. The complete coding sequence of one strain, K056/2008/TUR, was also generated, and molecular analysis of this strain provided further evidence of ongoing viral evolution and genetic diversity in Türkiye. Overall, these findings provide new insights into the prevalence, genetic diversity, and predicted immunogenic features of BKoVs in calves in Türkiye, supporting further studies on its pathogenesis, diagnostics, and antigenic properties. Full article

The 2019 coronavirus disease pandemic (COVID-19) showed how genetic mutations can alter coronavirus characteristics. However, the evolution of livestock coronaviruses remains understudied. We analyzed 15 bovine coronavirus (BCoV), three porcine hemagglutinating encephalomyelitis virus (PHEV) and 18 porcine respiratory coronavirus (PRCV) isolates, mainly from Belgian livestock collected between 2020 and 2023. Spike gene phylogenetic analysis showed nucleotide substitution rates comparable between BCoV and PRCV, while PHEV appeared slower. Unlike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), synonymous substitutions were preferred, limiting amino acid variation across decades in the animal coronaviruses. Virus neutralization assays with swine antisera indicated minimal antigenic change in PHEV and PRCV. Recent BCoV isolates showed antigenic divergence from the classical Mebus vaccine strain. The impact of this divergence on vaccine efficacy may warrant further research. Our findings underscore the need for periodic surveillance, as changes in surface proteins may affect pathogenicity, tissue tropism, host range and vaccine efficacy. Full article

To evaluate the diagnostic performance and clinicopathologic relevance of a multi-locus circulating tumor DNA methylation assay. In this prospective, single-center, case-control exploratory study, we enrolled 35 patients with colorectal cancer undergoing surgery and 57 healthy controls undergoing screening colonoscopy at the Asan Medical Center, Seoul, Republic of Korea between July 2024 and January 2025. Peripheral blood was collected before surgery or colonoscopy, and circulating tumor DNA methylation was analyzed using a multi-locus panel targeting Septin9, IKZF1, BCAT1, Septin9-2, BCAN, and VAV3. The main outcomes were test accuracy (sensitivity, specificity, and area under the curve [AUC]) and associations between methylation marker positivity and clinicopathologic features. Circulating tumor DNA was positive in 74.3% of the patients and 12.3% of controls, yielding a sensitivity of 74.3%, specificity of 87.7%, and an AUC of 0.837, whereas serum carcinoembryonic antigen exhibited lower sensitivity (25.7%). Sensitivity in stage I disease was limited (36.4%). Circulating tumor DNA-positive tumors were larger (5.7 cm vs. 2.2 cm, p < 0.001) and had more advanced T and N stages. The number of positive markers increased with pathologic stage (p = 0.003). Individual marker analysis revealed that BCAT1, Septin9-2, and VAV3 were associated with higher T stage, whereas BCAN positivity was linked to nodal metastasis. The six-marker circulating tumor DNA methylation assay demonstrated acceptable diagnostic accuracy, with multi-locus patterns associated with tumor burden and invasive features. However, sensitivity for early-stage disease was limited. The assay may serve as a complementary tool for screening and risk stratification. Full article

Dengue virus (DENV), which comprises four antigenically distinct serotypes (DENV-1 to DENV-4), remains a major global public health concern and continues to expand geographically; however, the structural features of the viral genome remain incompletely understood. Although G-quadruplexes (G4s) have previously been reported in coding regions of DENV, their presence within the 3′ untranslated region (3′ UTR) has not been experimentally characterized. Here, we focused on selected guanine-rich motifs within the 3′ UTRs of DENV-1 to DENV-4 and investigated their ability to form RNA G4 structures. Using bioinformatic analysis, we identified comparable G-rich regions in the 3′ UTRs of the four serotypes, with serotype-dependent differences in conservation. We then examined the propensity of the selected putative quadruplex-forming sequences (PQSs) to adopt G4 structures using circular dichroism spectroscopy, UV melting analysis, ¹H NMR spectroscopy, ligand-binding analysis, and reverse transcription stop (RT-stop) assays. Our results provided in vitro evidence that the 3′ UTR oligonucleotides from DENV-1 to DENV-4 are capable of forming ligand-responsive G4 structures, with serotype-dependent differences in conservation, stability, and conformational homogeneity. In addition, reverse transcription (RT)-stop analysis revealed ligand-dependent arrest at the corresponding PQS sites in the presence of the G4 ligand 6OTD, which stabilizes G4 structures. These findings suggest the DENV 3′ UTR as an additional source of ligand-responsive RNA G4-forming elements and support future studies on their possible roles in DENV RNA regulation. Full article

Background: Lipopolysaccharide (LPS) functions as a Toll-like receptor 4 (TLR4) agonist that triggers innate immunity; however, structural variations between pathogenic and commensal bacteria distinctly influence its immunostimulatory profile. This study evaluated the immunostimulatory activity of LPS derived from the commensal bacterium Hafnia alvei and explored its potential as an exploratory vaccine adjuvant. Methods: Cytokine induction was evaluated in immune cells across diverse host species, and receptor activation was assessed via reporter assays. To investigate in vivo immunogenicity and preliminary tolerability, H. alvei LPS was formulated into a prototype oil-in-water (O/W) emulsion utilizing ovalbumin (OVA) as a model antigen. Results: LPS from H. alvei strain BA2000346 exhibited immunostimulatory activity comparable to that of Escherichia coli, while inducing greater TNF-α expression than pathogenic Salmonella and Pseudomonas strains. Distinct from E. coli LPS, it demonstrated the capacity to activate both TLR4 and the mannose-recognizing Dectin-2 receptor in reporter systems. This cytokine induction was consistent across various strains and host species. Furthermore, the prototype O/W emulsion formulation enhanced antigen-specific humoral and cellular immune responses while demonstrating preliminary tolerability based on body-weight monitoring and visual clinical observation. Conclusions: H. alvei-derived LPS exhibits TLR4 and Dectin-2 agonist activity in vitro. When synergized with an O/W emulsion delivery system, it provides a preliminary indication of cross-species stimulatory potential and supports further investigation as a hypothesis-generating platform for future vaccine adjuvant development. Full article

Immune-mediated neutropenias comprise a heterogeneous group of disorders characterized by antibody-mediated destruction of neutrophils, in which the detection of anti-neutrophil antibodies remains a significant diagnostic challenge. Human neutrophil antigens (HNAs) are key targets in both autoimmune and alloimmune conditions, and their identification requires an integrated laboratory approach combining serological assays, HNA genotyping, and clinical evaluation. However, variability in assay sensitivity, the presence of low-titer or conformationally dependent antibodies, and interference from anti-HLA antibodies may lead to inconclusive or misleading results. This review summarizes the immunological mechanisms underlying anti-HNA antibody-mediated neutropenia and critically evaluates current laboratory methods, including cell-based and bead-based assays. The role of HNA genotyping in supporting antibody identification and improving diagnostic accuracy is also discussed. In addition, we highlight the importance of interpreting serological findings according to antibody specificity and clinical context. An integrated and multidisciplinary diagnostic approach is essential to ensure accurate diagnosis and appropriate clinical management, while emerging technologies may further improve antibody detection in the future. Full article

Viral hemorrhagic fevers (VHFs) are severe infectious diseases caused by RNA viruses of the families Arenaviridae, Filoviridae, Flaviviridae, and Hantaviridae, characterized by high morbidity, significant case fatality rates, and frequent diagnostic uncertainty in early disease stages. For military medical services, timely clinical recognition and laboratory confirmation are essential to guide patient management, prevent nosocomial transmission, and maintain operational continuity, particularly in endemic or resource-limited deployment settings. This review critically examines current diagnostic approaches to VHF-causative agents, emphasizing their use in clinical and field medical settings. The diagnostic process, from exposure through specimen collection, laboratory testing, and result interpretation is analyzed, including the use of molecular, serological, and antigen-based assays. Particular attention is given to deployable diagnostic platforms and their role in bridging the gap between frontline clinical suspicion and definitive laboratory confirmation. Biosafety requirements and infection prevention measures are discussed as integral components of clinical diagnostic workflows, aligned with guidance from the World Health Organization and the Centers for Disease Control and Prevention. Comparative analyses of virus-specific diagnostic timelines and laboratory requirements are presented to support differential diagnosis and clinical decision-making. Emerging technologies, including rapid molecular assays and genomic methods, are evaluated for their potential to improve early diagnosis and patient outcomes. This review highlights the central role of diagnostic readiness in clinical management of the VHFs and provides evidence-based considerations to support military clinicians facing high-risk febrile illnesses in operational environments. Full article

African swine fever in both domestic and wild pig populations is caused by the extremely infectious African swine fever virus (ASFV). It seriously endangers biodiversity and results in large financial losses for the worldwide pork sector. The major capsid protein p72 is molecularly chaperoned by the ASFV pB602L protein, which is essential to viral assembly. Furthermore, as a nonstructural protein expressed at late stages of infection, pB602L induces a distinct antibody response that may complement existing serological assays based on structural proteins. Given its strong immunogenicity, pB602L represents a promising antigen for developing supplementary diagnostic tools for African swine fever (ASF). In this study, we successfully generated and separated the ASFV pB602L protein, and we verified its responsiveness using serum from pigs infected with ASFV. Additionally, we produced four monoclonal antibody-specific hybridoma cell lines that targeted the pB602L protein exclusively. These cell lines demonstrated high immunoreactivity and responsiveness toward ASFV pB602L. These results highlight the potential enhancement of diagnostic skills. We have detected two previously unknown linear B-cell epitopes (¹³⁸TIDSFL¹⁴³ and ¹⁶⁴TNVDTC¹⁶⁹) using overlapping peptide and truncated protein fragment analysis. Due to their high degree of conservation across various ASFV strains, these epitopes offer trustworthy candidates for the creation of particular diagnostic instruments. This study expands the known ASFV antigenic repertoire by systematically mapping immunodominant epitopes of pB602L. The identified epitopes provide potential molecular targets for the rational design of multi-epitope subunit vaccines. Full article

Cross-presentation of exogenous antigens by dendritic cells (DCs) relies on the cytosolic pathway, enabling proteasomal processing and subsequent loading of antigenic peptides onto major histocompatibility complex class I (MHC-I) molecules. Although this pathway is central to CD8⁺ T-cell activation, the molecular mechanisms that regulate intracellular antigen processing and redistribution during cross-presentation remain incompletely defined. In this study, we investigated the contribution of the large-pore channel Pannexin-1 (Panx1) to antigen handling during cross-presentation. Using confocal microscopy and quantitative image analysis in granulocyte–macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4)-derived inflammatory bone marrow-derived dendritic cell (BMDC)-like cellsexposed to ovalbumin (OVA)–Alexa Fluor 488, we observed time-dependent changes in intracellular antigen distribution that were altered upon pharmacological inhibition of Panx1 with the blocking peptide 10Panx1. In parallel, functional assays revealed that Panx1 inhibition significantly reduced SIINFEKL peptide-dependentactivation of B3Z CD8⁺ T-cell hybridomas following pulsing with full-length OVA. Similar effects were observed in the cross-presentation-competent MUTU1940 dendritic cell line. Importantly, Panx1 inhibition did not significantly affect dendritic-cell viability or LPS-induced activation under the experimental conditions tested. In contrast, pharmacological inhibition or genetic deficiency of P2X7 receptor (P2X7) did not produce comparable reductions in cross-presentation, and combined inhibition did not result in additive effects under the experimental conditions tested. Together, these findings provide functional evidence supporting a role for Panx1 in regulating intracellular antigen redistribution associated with cross-presentation. While not establishing direct genetic causality, our data identify Panx1 as a modulatory component influencing antigen-processing events that culminate in CD8⁺ T-cell activation, thereby expanding the current framework of intracellular antigen-processing mechanisms involved in dendritic-cell-mediated cross-presentation. Full article

Background and Objectives: Blood group antigens are well known for their importance in transfusion medicine and transplant compatibility; however, their biological role extends beyond these functions and includes associations with the risk of several diseases. In this study, we investigated the relationship between ABO blood groups and the circulating levels of 73 different molecules. Patients and Methods: Fifty-six healthy donors were enrolled, including 24 individuals with blood group O, 19 with blood group A, and 13 with blood group B. Blood samples were collected and analyzed in a single laboratory using Luminex fluorescent bead-based assay panels to determine the concentrations of 73 circulating molecules. Depending on data distribution, ANOVA or Kruskal–Wallis tests and Student’s t-test or Kolmogorov–Smirnov tests were applied to identify significant differences among groups. Associations were further assessed by binary logistic regression analysis. Results: Subjects with blood group A showed significantly higher circulating levels of IL-1R1, IL-13, IL-23, PDGF-BB, VEGF-A, VEGF-D, soluble VEGF-R2 (KDR), soluble VEGF-R3 (FLT-4), VLA-4, CD141, MMP-1, syndecan-1 (SDC-1), and mannose-binding lectin (MBL) compared with the other blood groups. In contrast, individuals with blood group B exhibited significantly higher levels of IL-22, IL-23, PDGF-BB, CD62P (P-selectin), and amyloid β1–42. Several significant associations were identified by logistic regression analysis. Conclusions: Our findings indicate that ABO blood groups are associated with distinct circulating molecular profiles, supporting the existence of biological differences that may contribute to variations in disease susceptibility among individuals with different blood types. Nevertheless, given the exploratory’s nature and limited sample size of this study, further investigations are required to validate these findings, confirm the observed associations, and clarify their potential clinical implications. Full article

The genetic and antigenic diversity of H5Nx HPAI Gs/GD lineage continues to be a great challenge facing conventional inactivated vaccines. To overcome this challenge, a recombinant herpes virus of turkey (rHVT) vaccine expressing the viral protein 2 (VP2) of infectious bursal disease (IBD) and H5, rHVT+IBD+H5, was developed using computationally optimized broadly reactive antigen (COBRA) technology. In the current study, the protective efficacy of a commercially available vector trivalent vaccine rHVT+IBD+H5 using COBRA technology was assessed. A total of 120 commercial broilers were divided equally into six groups (G1B–G6B). The chickens in G1B–G3B were challenged with the most recent circulating HPAI-H5N1 clade 2.3.4.4.b Egyptian isolate (GenBank accession No. OQ933425) at 28 days old (DO), while the chickens in G4B and G5B were kept as vaccinated (as G1B and G2B, respectively) and non-challenged, and G6B was the non-vaccinated non-challenged group. In G1B, the chickens were vaccinated with Vaxxitek^® rHVT+IBD+H5 at 1 DO and boostered with a commercially available subunit Baculovirus bivalent inactivated H5+ND (Volvac^® B.E.S.T AI+ND) at 10 DO and had a 100% survival rate. The standalone vaccinated chicken G2B, using rHVT+IBD+H5 at 1 DO, had a highly significant survival rate (90%) vs. 0% (100% mortality) in the non-vaccinated challenged control, G3B. All the vaccinated groups had higher seroconversion at 45 DO especially using H5-coated antigen plates for the enzyme-linked immunosorbent assay (ELISA) test. The viral shedding titers and time were evaluated using a quantitative real-time polymerase chain reaction (RT-qPCR) in the collected oropharyngeal and cloacal swabs at 3, 5, 7, and 10 days post-challenge (DPC). In conclusion, vaccination with rHVT+IBD+H5 either as a standalone or when boostered with subunit Baculovirus bivalent inactivated ND+H5 resulted in 90 and 100% protection, respectively, without significant difference in the quantity and duration of viral shedding between both groups against HPAI-H5N1 clade 2.3.4.4.b experimental challenge in broilers. Full article

Patients with myeloproliferative neoplasms (MPN) are at increased risk of herpes zoster, particularly during Janus kinase inhibitor (JAKi) therapy, yet the immunogenicity of recombinant zoster vaccine (RZV) in this setting remains incompletely characterized. We performed a prospective pilot translational study including 18 patients with MPN and a small age-matched healthy donor group (n = 4, descriptive reference only). Samples were collected at baseline, 21 days after the first dose, and 21 days after the second dose. Humoral response was assessed by anti-varicella-zoster virus (VZV) immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), whereas antigen-specific cellular responses were evaluated after ex vivo stimulation with recombinant VZV glycoprotein E followed by flow cytometry and cytokine quantification. IgG levels increased over time in MPN patients, while cellular responses remained limited, heterogeneous, and not consistently enhanced. Cytokine production was low and variable across time points. Overall, RZV in MPN under JAKi was associated with detectable humoral responses but limited cellular activation, supporting an apparent discordance between humoral and cellular immune readouts under the experimental conditions used. Full article

Spotted fever group (SFG) rickettsioses are emerging zoonotic diseases of increasing relevance in Latin America, yet the specific species involved in human infections remain poorly defined in many endemic regions. This study aimed to determine the most probable antigen among SFG-seroreactive febrile patients from Villeta, Colombia. A panel of 25 convalescent-phase serum samples previously identified as positive for SFG Rickettsia spp. antibodies was analyzed by indirect immunofluorescence assay using antigens of Rickettsia rickettsii, R. amblyommatis and R. parkeri. Antibody titers were compared to identify differential seroreactivity patterns. Overall, 44% (11/25) of the samples showed differential antibody titers against one of the tested antigens. Among these, nine (36%) exhibited higher titers to R. parkeri and two (8%) to R. amblyommatis, while none showed exclusive reactivity to R. rickettsii. The remaining 56% (14/25) presented similar titers across antigens, consistent with indeterminate or cross-reactive SFG responses. Antibody titers ranged from 1:128 to 1:4096, with R. parkeri showing the strongest reactivity. These findings suggest R. parkeri or a highly related Rickettsia species as the predominant probable antigen in Villeta, highlighting its potential role in mild rickettsial infections and emphasizing the need for eco-epidemiological studies to identify local vectors and reservoirs. Full article

Background/Objectives: Latent tuberculosis infection (LTBI) is a persistent immune response to Mycobacterium tuberculosis antigens in the absence of clinically active tuberculosis. It is now established that progression from LTBI to active tuberculosis is directly associated with immune dysregulation, which frequently occurs in immune-mediated diseases requiring treatment with immunosuppressive agents. The aim of this study was to identify LTBI in patients with rheumatological diseases receiving immunosuppressive therapy using contemporary immunodiagnostic methods. Materials and Methods: A retrospective, prospective, group-control study was conducted, analyzing the results of immunodiagnostics in patients with rheumatological diseases on immunosuppressive therapy and without established contact with tuberculosis patients (n = 44; main group). The control group consisted of healthy individuals (n = 51) with no history of tuberculosis contact, clinical or radiological manifestations of the disease, or signs of chronic pathology exacerbation. Both groups were predominantly female (72.7% in the main group and 62.8% in the control group). The mean age in the patient group was 49.1 years (95% CI [44.77; 53.43]). Rheumatoid arthritis was diagnosed in 29.6% (13) of patients (95% CI [16.06; 43.03]). Articular syndrome was observed in at least 72.7% (32) of patients (95% CI [59.57; 85.89]). In 54.6% (24) of cases (95% CI [39.83; 69.26]), patients received biologic immunosuppressive therapy as basic treatment. In 15.9% (7) of cases (95% CI [5.10; 26.72]), patients received conventional synthetic disease-modifying antirheumatic drugs (DMARDs). Of these, 71.43% (5) (95% CI [35.24; 92.44]) underwent comprehensive examination to exclude active tuberculosis prior to biologic therapy initiation. For immunodiagnostics, all subjects underwent an interferon-gamma release assay (IGRA) and/or testing with a recombinant tuberculosis antigen (ATR) sample, with dynamic assessment of test results. All patients with positive immunodiagnostic results underwent multidetector computed tomography of the chest organs. The level of LTBI in the comparison groups was defined as the percentage of positive immunological test results at a significance level of p < 0.05. Statistical data processing was performed using Microsoft Excel 2019. Results: In the main group, positive immunodiagnostic results were recorded in 20.5% (9) of cases (95% CI [8.54; 32.37]), which is significantly higher than in the control group of healthy individuals (5.8% (3), 95% CI [1.41; 16.54]). This reflects statistically significant differences in immunological test results between groups receiving and not receiving immunosuppressive therapy (χ² = 4.545, p = 0.034). Dynamic evaluation of the ATR sample revealed positive results in 21.7% (5) of cases (95% CI [4.88; 38.60]), with four out of five patients demonstrating positive conversion. In the third assessment, positivity was observed in 33.33% (5) of cases (95% CI [9.48; 57.19]), which was higher than in the first (χ² = 1.025, p = 0.312) and second assessments (χ² = 0.629, p = 0.428), although these differences were not statistically significant. Notably, in two out of five patients, the ATR test result changed from negative to positive. Conclusions: In patients with rheumatological diseases receiving immunosuppressive therapy, LTBI was detected in 20.5%, which is significantly higher than in healthy individuals (5.8%, p = 0.034). Furthermore, there was an increase in the proportion of positive tests over time (up to 21.7% and 33.3% on immunotherapy), suggesting an increasing risk of progression to active tuberculosis infection. Full article

During construction of the Baku–Tbilisi–Kars railroad between Kars City in Türkiye and Tbilisi in Georgia, blasting operations in an anthrax-endemic region disrupted a burial pit containing carcasses of cattle that had died of anthrax. Railroad workers expressed concerns that release of this material could result in them developing anthrax infection. We therefore undertook a seroprevalence study six months later to seek evidence of exposure to Bacillus anthracis spores. We used an optimised Enzyme-Linked Immunosorbent Assay (ELISA) to screen serum for antigen-specific IgG antibodies to the anthrax toxin subunits Protective Antigen (PA) and Lethal Factor (LF). Stepwise linear regressions and t-tests were performed to compare results from railroad workers (n = 64) with a group of long-term Kars City residents (urban dwellers, n = 16), who had no history of possible contact with anthrax antigens. Anti-PA IgG concentrations were higher (p = 0.038) in railroad workers than in urban dwellers, but anti-LF IgG concentrations did not differ (p = 0.932) between the two groups. The anti-PA response is known to be dominant, and the difference was small. The lack of LF response did not preclude an antibody response to B. anthracis. Following the blasting operations, no cases of anthrax infection occurred in either railroad workers or villagers living nearby, suggesting that the spore exposure (evidenced by higher antibody titres) was at levels insufficient to initiate clinical infection. The elevated PA-specific antibody responses in railroad workers compared with urban dwellers might be consistent with the former having had previous subclinical exposure to B. anthracis. In anthrax-endemic regions, therefore, construction activities that involve blasting or large-scale excavation may pose risks of occupational exposure to Bacillus anthracis spores. Full article