Aneta Pluta

Bovine leukemia virus (BLV) is a deltaretrovirus that establishes lifelong infection in cattle and remains highly prevalent in dairy herds. This study evaluated associations between maternal BLV infection markers, daughter BoLA-DRB3 variation, and BLV outcomes in dairy youngstock. We studied 201 dam–daughter pairs from five dairy farms. BLV status was assessed using ELISA and quantitative PCR (qPCR); maternal qPCR results were available for 126 dams, and daughters were sampled longitudinally during the youngstock period. BoLA-DRB3 genotypes were determined in 199 daughters, and genotype-level epitope breadth was defined as the non-redundant union of predicted BLV Gag epitopes across both alleles. BLV proviral DNA was detected in 33 of 200 daughters (16.5%) in the qPCR outcome used for the main analysis. Among 38 daughters with BLV proviral DNA detected at least once, first detection occurred preweaning in 2, prebreeding in 24, and post-breeding in 12. Maternal ELISA positivity was associated with higher odds of daughter BLV proviral DNA detection (OR 2.74, 95% CI 1.10–6.83), whereas maternal PVL showed a positive but imprecise association (OR 1.94, 95% CI 0.89–4.22). Greater predicted epitope breadth was nominally associated with lower odds of BLV proviral DNA detection (OR 0.76, 95% CI 0.60–0.97; q = 0.162) and BLV seropositivity (OR 0.80, 95% CI 0.65–0.98; q = 0.162), but neither association remained significant after correction for multiple testing. BoLA-DRB3*014:01:01 carriers also had lower observed frequencies of BLV proviral DNA detection and BLV seropositivity. Overall, maternal infection markers were associated with later BLV outcomes in daughters, whereas the BoLA-DRB3 findings provide suggestive evidence of host immunogenetic heterogeneity.

Bovine leukemia virus (BLV) encodes a cluster of viral microRNAs (miRNAs) that remain abundantly expressed during latency, when viral protein production is restricted. Naturally occurring single-nucleotide polymorphisms (SNPs) within this locus are common, yet their functional consequences for miRNA output and host gene regulation remain poorly defined. Peripheral blood leukocytes from 53 naturally BLV-infected cattle were analyzed, and the 554-nt BLV miRNA locus was amplified and sequenced. Field-derived variants containing mutations within RNA polymerase III promoter motifs, seed regions, and termination signals were selected for functional evaluation. Reference and variant loci were co-expressed with a BLVΔ-miRNA infectious clone in HEK293T cells under controlled conditions. Mature miRNA levels were quantified by stem-loop reverse-transcription quantitative polymerase chain reaction (RT–qPCR), and global host transcriptional responses were assessed using oligonucleotide microarrays. Predicted miRNA targets were identified using bioinformatic analyses. Sequence analysis identified 84 polymorphic sites, with a substantial proportion mapping to Pol III regulatory elements and seed regions. Variant loci displayed altered accumulation of selected mature miRNAs and shifts in predicted seed-dependent target repertoires. Transcriptome profiling revealed variant-associated modulation of innate immune-signaling components, interferon-responsive genes, antigen-presentation pathways, tumor-suppressor networks, and extracellular matrix-related processes. Enrichment analysis demonstrated a statistically significant overlap between predicted miRNA targets and downregulated transcripts. Natural polymorphisms within the BLV miRNA cluster modulate miRNA expression and are associated with distinct host regulatory signatures in a controlled experimental system. These findings suggest that sequence variation in the viral miRNA locus may contribute to differential host–virus interactions and influence mechanisms supporting viral persistence.

Bovine leukemia virus (BLV) is a cell‐associated retrovirus of cattle transmitted mainly through the transfer of infected lymphocytes, but the possible role of hematophagous insects in its epidemiology remains incompletely understood. This study investigated BLV DNA in field‐collected Culicoides biting midges and Stomoxys calcitrans from cattle farms in Poland. Insects were collected on four farms in northeastern Poland. Culicoides spp. were identified morphologically, classified by gonotrophic status, and pooled by species and status, whereas S. calcitrans were analyzed individually. DNA quality was verified by PCR targeting insect markers. Bovine host DNA was detected by PCR targeting mitochondrial cytochrome b in Culicoides and prepronociceptin (PNOC) in S. calcitrans . BLV proviral DNA was detected by TaqMan real‐time PCR targeting the pol gene, followed by nested PCR and sequencing of a 400 bp fragment of the env gene. BLV DNA was detected in 20 of 138 Culicoides pools (14.5%). BLV copy numbers ranged from 2 to 19,000 copies per 500 ng of total DNA. All BLV‐positive Culicoides pools were also positive for bovine cytochrome b DNA. Among the pol ‐positive pools, 18 were positive in nested env PCR, and 15 yielded high‐quality sequences. One S. calcitrans specimen was also positive in both BLV assays. Sequences obtained from insects were identical to those from infected cattle, and phylogenetic analysis assigned them to genotypes G4 and G7, the predominant BLV genotypes circulating in Poland. These findings provide field‐based molecular evidence that Culicoides biting midges and S. calcitrans can carry bovine blood and BLV DNA under natural farm conditions, supporting their possible role in the mechanical transfer of BLV between cattle.

The common carp Cyprinus carpio L. is a cornerstone aquaculture species, yet transcriptome interpretation is complicated by its paleotetraploid genome and extensive alternative splicing. A species-tailored oligonucleotide microarray was developed to deliver reproducible, gene-level expression profiling. Probe design was anchored to the SPL01 reference and implemented on an Agilent platform using a gene-level strategy that collapsed transcripts to genes, selected the longest isoform, and placed 3′-anchored 60-mer probes. The workflow incorporated embedded technical controls and a standardized two-color pipeline to ensure stable measurements across arrays. Baseline functional organization of the head kidney was defined using 614 C. carpio L. -Danio rerio orthologs and complementary enrichment tools. Coherent signatures emerged for hemoglobin-mediated oxygen transport, heme and porphyrin metabolism, antioxidant defense driven by peroxiredoxin and thioredoxin systems, including hydrogen peroxide detoxification, protease regulation through SERPIN, SPINK, and WFDC families, and elements of innate and humoral immunity. Targets bearing c-Myc motifs showed a modest positive bias consistent with ongoing hematopoiesis. These resolved baseline modules provide a reference against which infection- or exposure-induced programs such as interferon-stimulated genes, chemokines and chemotaxis, complement activation, and degranulation can be detected and quantified. The platform complements RNA-seq by offering cost-efficient, rapid, and comparable measurements suited to large cohorts and longitudinal designs. Anticipated applications include host–pathogen studies for viral and bacterial agents and the assessment of chemical contaminants in aquaculture surveillance, supporting standardized, cross-study decision-making in research and health monitoring.

Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that induces enzootic bovine leukosis. A defining feature of BLV is its viral miRNA cluster, which is transcribed atypically by RNA polymerase III via internal type 2 promoters rather than through the canonical Pol II pathway. These miRNAs accumulate to high levels within infected lymphocytes and can alter expression of a variety of host genes involved in lymphocyte proliferation and impose leukemogenic processes. Here, we present a comprehensive in silico characterization of new A-box and B-box promoter motifs within the BLV miRNA-coding region. As the first step, a taxonomically diverse dataset of small non-coding RNAs (tRNAs, SINEs, and other ncRNAs) was assembled to derive position-weight matrices and corresponding IUPAC consensus sequences for type 2 internal Pol III promoter motifs. Using these models, all available BLV miRNA cluster sequences were scanned to identify and map A-box-like and B-box-like elements and to reconstruct the underlying promoter architecture. Our analyses reveal a noncanonical BLV promoter organization: overlapping degenerate A-box variants—most frequently three distinct elements—reside within the pre-miRNA hairpin region, whereas B-box elements were positioned downstream of the Pol III termination signal, effectively excluded from the mature transcript. Despite motif degeneration, critical nucleotide positions remained strongly conserved, indicating evolutionary pressure to preserve Pol III recruitment while accommodating viral genome constraints. These findings fill a crucial gap in understanding of BLV Pol III promoter architecture and provide a foundation for future studies on how unconventional promoter configurations regulate viral miRNA expression and virus–host interactions.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

We have characterized the intrahost genetic variation in the bovine leukemia virus (BLV) by examining 16 BLV isolates originating from the Western Siberia–Tyumen and South Ural–Chelyabinsk regions of Russia. Our research focused on determining the genetic composition of an 804 bp fragment of the BLV env gene, encoding for the entire gp51 protein. The results provide the first indication of the quasi-species genetic nature of BLV infection and its relevance for genome-level variation. Furthermore, this is the first phylogenetic evidence for the existence of a dual infection with BLV strains belonging to different genotypes within the same host: G4 and G7. We identified eight cases of recombination between these two BLV genotypes. The detection of quasi-species with cases of dual infection and recombination indicated a higher potential of BLV for genetic variability at the intra-host level than was previously considered.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region– positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Characterization of the global genetic diversity of the bovine leukemia virus (BLV) is an ongoing international research effort. Up to now BLV sequences have been classified into eleven distinct genotypes. Although BLV genotyping and molecular analysis of field isolates were reported in many countries, there is no report describing BLV genotypes present in cattle from Pakistan. In this study we examined 27 env gene sequences from BLV-infected cattle coming from four farms located in Khyber Pakhtunkwa, Gilgit Baltisan and Punjab provinces. Phylogenetic analyses revealed the classification of Pakistani sequences into genotypes G1 and G6. The alignment with the FLK-BLV sequence revealed the presence of 45 mutations, namely, seven in genotype G1 and 33 in genotype G6. Five mutations were found in both, G1 and G6 genotypes. Twelve amino acid substitutions were found in the analyzed sequences, of which only one P264S was specific for sequences from Pakistan. Furthermore, a certain degree of nucleotide heterogeneity was identified by NGS. These results highlight the need for further study on the importance of genetic variability of BLV, especially in the context of its pathogenicity and potential effect on serological detection.

Bovine Leukemia Virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), a lymphoproliferative disease of the bovine species. In BLV-infected cells, the long terminal repeat (LTR), the viral Tax protein and viral miRNAs promote viral and cell proliferation as well as tumorigenesis. Although their respective roles are decisive in BLV biology, little is known about the genetic sequence variation of these parts of the BLV genome and their impact on disease outcome. Therefore, the objective of this study was to assess the relationship between disease progression and sequence variation of the BLV Tax, miRNA and LTR regions in infected animals displaying either low or high levels of persistent lymphocytosis (PL). A statistically significant association was observed between the A(+187)C polymorphism in the downstream activator sequence (DAS) region in LTR (p-value = 0.00737) and high lymphocytosis. Our study also showed that the mutation A(−4)G in the CAP site occurred in 70% of isolates with low PL and was not found in the high PL group. Conversely, the mutations G(−133)A/C in CRE2 (46.7%), C(+160)T in DAS (30%) and A(310)del in BLV-mir-B4-5p, A(357)G in BLV-mir-B4-3p, A(462)G in BLV-mir-B5-5p, and GA(497–498)AG in BLV-mir-B5-3p (26.5%) were often seen in isolates with high PL and did not occur in the low PL group. In conclusion, we found several significant polymorphisms among BLV genomic sequences in Russia that would explain a progression towards higher or lower lymphoproliferation. The data presented in this article enabled the classification between two different genotypes; however, clear association between genotypes and the PL development was not found.