Characterization and application of recombinant Bovine Leukemia Virus Env protein.

The Bovine Leukemia Virus (BLV) Envelope (Env) glycoprotein complex is instrumental in viral infectivity and shapes the host's immune response. This study presents the production and characterization of a soluble furin-mutated BLV Env ectodomain (sBLV-EnvFm) expressed in a stable S2 insect cell line. We purified a 63 kDa soluble protein, corresponding to the monomeric sBLV-EnvFm, which predominantly presented oligomannose and paucimannose N-glycans, with a high content of core fucose structures. Our results demonstrate that our recombinant protein can be recognized from specific antibodies in BLV infected cattle, suggesting its potential as a powerful diagnostic tool. Moreover, the robust humoral immune response it elicited in mice shows its potential contribution to the development of subunit-based vaccines against BLV.

Bovine Leukemia Virus molecular detection and associated factors among dairy herd workers in Antioquia, Colombia.

The Bovine Leukemia Virus (BLV) affects mainly cattle, is transmitted by exposure to contaminated biological fluids, and generates lymphomas in 5 % of infected animals. The zoonotic potential of BLV has been studied, and it is currently unknown if it circulates in human workers on dairy herds in Antioquia. Objective: To determine the frequency of BLV detection, the genotypes of the virus, and the factors associated with its detection in workers for dairy herds in Antioquia, Colombia. Through a cross-sectional study in 51 dairy herds, 164 adults were recruited. A peripheral blood sample was collected from each participant for molecular detection of the BLV env and tax genes, and associated factors were explored through bivariate and multivariate mixed Poisson model analyses. The analysis showed that 82 % (134/164) of the participants were men, with an average age of 40. Using qPCR, the constitutive gene GAPDH was amplified to evaluate the presence of amplification inhibitors in the DNA samples. Using nested PCR, the amplification of the env viral gene was obtained in 13 % (22/164) of the total samples analyzed, while all the samples tested negative for tax. The amplicons of the env gene were sequenced, and the identity compatible with BLV was verified by BLAST analysis (NCBI). Using molecular phylogeny analysis, based on maximum likelihood and haplotype network analysis, it was identified that BLV genotype 1 is present in the evaluated population. 16 % (26/164) of the participants reported having ever had an accident with surgical material during work with cattle; this variable was associated with BLV positivity even after adjusting for other variables (PRa =2.70, 95 % CI= 1.01- 7.21). Considering that other studies have reported the circulation of BLV genotype 1 in cattle from this same region and the present report in humans from dairy herds, the results suggest a possible zoonotic transmission of BLV genotype 1 in Antioquia, reinforcing the need to continue investigating to determine the potential role of this virus as an etiological agent of disease in livestock farmers in the department.

Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Monoclonal proliferation of B-cells with two integration sites of bovine leukemia virus proviral DNA in cattle with enzootic bovine leukosis.

The present study analyzed B-cell clonality and bovine leukemia virus (BLV) provirus integration sites in cattle with enzootic bovine leukosis (EBL) having BLV proviral copy numbers less or greater than the number of bovine nucleated cells. EBL cattle with BLV copy numbers less than the number of bovine nucleated cells showed monoclonal and biclonal proliferation of B-cells with one BLV provirus integration site. On the other hand, EBL cattle with BLV copy numbers greater than the number of bovine nucleated cells showed monoclonal proliferation of B-cells with two BLV provirus integration sites. These results suggest that superinfection of BLV can occur in EBL cattle.

Chimeric provirus of bovine leukemia virus/SMAD family member 3 in cattle with enzootic bovine leukosis.

Bovine leukemia virus (BLV) is a member of the family Retroviridae that causes enzootic bovine leukemia (EBL). However, the association between BLV infection and EBL development remains unclear. In this study, we identified a BLV/SMAD3 chimeric provirus within CC2D2A intron 30 in monoclonal expanded malignant cells from a cow with EBL. The chimeric provirus harbored a spliced SMAD3 sequence composed of exons 3-9, encoding the short isoform protein, and the BLV-SMAD3 chimeric transcript was detectable in cattle with EBL. This is the first report of a BLV chimeric provirus that might be involved in EBL tumorigenesis.

Molecular analysis of the env, LTR, and pX regions of bovine leukemia virus in dairy cattle of Türkiye.

Bovine leukemia virus is a retrovirus that causes enzootic bovine leukosis and is associated with global economic losses in the livestock industry. The aim of this study was to investigate the genotype determination of BLVs from cattle housed in 6 different farms in Türkiye and the characterization of their LTR and pX (tax, rex, R3, and G4 gene) regions. For this purpose, blood samples from 48 cattle infected with BLV were used. The phylogenetic analysis based on the env gene sequences revealed that all BLVs were clustered in genotype 1 (G1), and the sequences of the LTR (n = 48) and the pX region (n = 33) of BLVs were obtained. Also, analysis of these nucleic acid and amino acid sequences allowed assessments similar to those reported in earlier studies to be relevant to transactivation and pathogenesis. This study reports the molecular analysis of the LTR and pX region of BLVs in Türkiye for the first time.

An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus.

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.

Enzootic bovine leukosis caused by bovine leukemia virus classified as Group C based on viral whole genome sequencing in a 23-month-old Holstein-Friesian heifer.

A 23-month-old Holstein-Friesian heifer presented with inactivity and diarrhea. On physical examination, no enlargement of superficial lymph nodes was observed. Hematological examination revealed lymphocytosis. The bovine leukemia virus (BLV) proviral load was 2,122 copies/10 ng DNA, and BLV was classified as Group C based on whole genome phylogenetic analysis. Monoclonal proliferation of B-cells and monoclonal integration of the BLV provirus in the bovine genome were detected by a clonality test of B-cells and inverse PCR, respectively. Although lymph nodes were not swollen at necropsy, histopathological examination revealed neoplastic lymphocyte proliferation in lymph nodes, which were immune positive for CD5 and CD20, and negative for CD3. The heifer was diagnosed with EBL caused by BLV classified as Group C.

Study of the proviral load levels and mRNA expression of cytokines in peripheral blood mononuclear cells and somatic milk cells in cattle with different BLV infection profiles.

The retrovirus bovine leukemia virus (BLV) might produce abnormal immune function, associated with susceptibility to developing other infectious diseases, including mastitis. This study aimed to determine the proviral load and cytokines gene expression in peripheral blood mononuclear cells (PMBC) and milk somatic cells (SC) in BLV-infected and non-infected cattle. Of 27 BLV-infected cows in PBMC, 17 (62.96%) had a high proviral load (HPL), and 10 (37.04%) had a low proviral load (LPL). All SC samples had low proviral load (LPL-SC). Higher IFN-γ and IL-10 expression, and lower IL-12 and IL-6 expression, were found in PBMC from BLV-infected compared to BLV non-infected cattle. Moreover, higher IFN-γ, IL-12, and IL-6 expression, and lower IL-10 expression were observed in cattle with LPL-PBMC compared to HPL-PBMC. In milk samples, lower IFN-γ and higher IL-12 mRNA expression were observed in LPL-SC compared to BLV non-infected cattle in SC. IL-10 and IL-6 expression mRNA was significantly lower in LPL-SC than in SC from BLV non-infected cattle. This study shows that milk SC maintains lower proviral load levels than PBMC. This first report on Th1 and Th2 cytokines expression levels in SC may be relevant to future control strategies for BLV infection, mastitis, and udder health management.

Low proviral load in the Kumamoto strain of Japanese Brown cattle infected with the bovine leukemia virus.

BACKGROUND: The Kumamoto strain of Japanese Brown (JBRK) cattle is a sub-breed of Wagyu and has a different genetic background than that of Japanese Black (JB) cattle. Bovine leukemia virus (BLV) is the pathogen causing enzootic bovine leukosis (EBL), the predominant type of bovine leukosis (BL). EBL is one of the most common bovine infectious diseases in dairy countries, including Japan. Some host genetic factors, including the bovine leukocyte antigen (BoLA)-DRB3 gene, have been associated with the proviral load (PVL) of BLV and/or onset of EBL. Here, we determined the number of BL cases by analyzing prefectural case records in detail. We measured the PVL of BLV-infected JBRK cattle and compared it with that obtained for other major breeds, JB and Holstein-Friesian (HF) cattle. Finally, the relationship between PVL levels and BoLA-DRB3 haplotypes was investigated in BLV-infected JBRK cattle. RESULTS: We determined the number of BL cases recorded over the past ten years in Kumamoto Prefecture by cattle breed. A limited number of BL cases was observed in JBRK cattle. The proportion of BL cases in the JBRK was lower than that in JB and HF. The PVL was significantly lower in BLV-infected JBRK cattle than that in the JB and HF breeds. Finally, in BLV-infected JBRK cattle, the PVL was not significantly affected by BoLA-DRB3 alleles and haplotypes. BoLA-DRB3 allelic frequency did not differ between BLV-infected JBRK cattle with low PVL and high PVL. CONCLUSIONS: To our knowledge, this is the first report showing that BL occurred less in the JBRK population of Kumamoto Prefecture. After BLV-infection, the PVL was significantly lower in JBRK cattle than that in JB and HF breeds. The genetic factors implicated in maintaining a low PVL have yet to be elucidated, but the BoLA-DRB3 haplotypes are likely not involved.

Exploring the presence of bovine leukemia virus among breast cancer tumors in a rural state.

PURPOSE: The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S. dairy herds. Associations between presence of BLV DNA in human mammary tissue and human breast cancer incidence have been reported. We sought to estimate the prevalence of BLV DNA in breast cancer tissue samples in a rural state with an active dairy industry. METHODS: We purified genomic DNA from 56 fresh-frozen breast cancer tissue samples (51 tumor samples, 5 samples representing adjacent normal breast tissue) banked between 2016 and 2019. Using nested PCR assays, multiple BLV tax sequence primers and primers for the long terminal repeat (LTR) were used to detect BLV DNA in tissue samples and known positive control samples, including the permanently infected fetal lamb kidney cell line (FLK-BLV) and blood from BLV positive cattle. RESULTS: The median age of patients from which samples were obtained at the time of treatment was 60 (40-93) and all were female. Ninety percent of patients had invasive ductal carcinoma. The majority were poorly differentiated (60%). On PCR assay, none of the tumor samples tested positive for BLV DNA, despite having consistent signals in positive controls. CONCLUSION: We did not find BLV DNA in fresh-frozen breast cancer tumors from patients presenting to a hospital in Vermont. Our findings suggest a low prevalence of BLV in our patient population and a need to reevaluate the association between BLV and human breast cancer.

A complex network of transcription factors and epigenetic regulators involved in bovine leukemia virus transcriptional regulation.

Bovine Leukemia Virus (BLV) is the etiological agent of enzootic bovine leukosis, a disease characterized by the neoplastic proliferation of B cells in cattle. While most European countries have introduced efficient eradication programs, BLV is still present worldwide and no treatment is available. A major feature of BLV infection is the viral latency, which enables the escape from the host immune system, the maintenance of a persistent infection and ultimately the tumoral development. BLV latency is a multifactorial phenomenon resulting in the silencing of viral genes due to genetic and epigenetic repressions of the viral promoter located in the 5' Long Terminal Repeat (5'LTR). However, viral miRNAs and antisense transcripts are expressed from two different proviral regions, respectively the miRNA cluster and the 3'LTR. These latter transcripts are expressed despite the viral latency affecting the 5'LTR and are increasingly considered to take part in tumoral development. In the present review, we provide a summary of the experimental evidence that has enabled to characterize the molecular mechanisms regulating each of the three BLV transcriptional units, either through cis-regulatory elements or through epigenetic modifications. Additionally, we describe the recently identified BLV miRNAs and antisense transcripts and their implications in BLV-induced tumorigenesis. Finally, we discuss the relevance of BLV as an experimental model for the closely related human T-lymphotropic virus HTLV-1.

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA.

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

Bovine leukemia virus encoded blv-miR-b4-3p microRNA is associated with reduced expression of anti-oncogenic gene in vivo.

Bovine leukemia virus (BLV) is a retrovirus that causes malignant B-cell lymphoma in up to ten-percent of infected cattle. To date, the mechanisms of BLV linked to malignant transformation remain elusive. Although BLV-encoded miRNAs have been associated with the regulation of different genes involved in oncogenic pathways, this association has not been evaluated in cattle naturally infected with BLV. The objective of this study was to determine the relative expression of BLV-encoded miRNA blv-miR-b4-3p, the host analogous miRNA bo-miR-29a and a couple of potential target mRNAs (HBP-1 and PXDN, with anti-tumorigenic function in B-cells), in cattle naturally infected with BLV compared to uninfected animals (control group). We observed that PXDN was significantly downregulated in BLV-infected cattle (P = 0.03). Considering the similar expression of endogenous bo-miR-29a in both animal groups, the downregulation of PXDN in BLV-naturally infected cattle could be linked to blv-miR-b4-3p expression in these animals. Knowing that PXDN is involved in anti-tumoral pathways in B-cells, the results presented here suggest that blv-miR-b4-3p might be involved in BLV tumorigenesis during natural infection with BLV in cattle.

Identifying Pathogen and Allele Type Simultaneously in a Single Well Using Droplet Digital PCR.

In the transmission control of chronic and untreatable livestock diseases such as bovine leukemia virus (BLV) infection, the removal of viral superspreaders is a fundamental approach. On the other hand, selective breeding of cattle with BLV-resistant capacity is also critical for reducing the viral damage to productivity by keeping infected cattle. To provide a way of measuring BLV proviral load (PVL) and identifying susceptible/resistant cattle simply and rapidly, we developed a fourplex droplet digital PCR method targeting the BLV pol gene, BLV-susceptible bovine major histocompatibility complex (BoLA)-DRB3*016:01 allele, resistant DRB3*009:02 allele, and housekeeping RPP30 gene (IPATS-BLV). IPATS-BLV successfully measured the percentage of BLV-infected cells and determined allele types precisely. Furthermore, it discriminated homozygous from heterozygous carriers. Using this method to determine the impact of carrying these alleles on the BLV PVL, we found DRB3*009:02-carrying cattle could suppress the PVL to a low or undetectable level, even with the presence of a susceptible heterozygous allele. Although the population of DRB3*016:01-carrying cattle showed significantly higher PVLs compared with cattle carrying other alleles, their individual PVLs were highly variable. Because of the simplicity and speed of this single-well assay, our method has the potential of being a suitable platform for the combined diagnosis of pathogen level and host biomarkers in other infectious diseases satisfying the two following characteristics of disease outcomes: (i) pathogen level acts as a critical maker of disease progression; and (ii) impactful disease-related host genetic biomarkers are already identified. IMPORTANCE While pathogen-level quantification is an important diagnostic of disease severity and transmissibility, disease-related host biomarkers are also useful in predicting outcomes in infectious diseases. In this study, we demonstrate that combined proviral load (PVL) and host biomarker diagnostics can be used to detect bovine leukemia virus (BLV) infection, which has a negative economic impact on the cattle industry. We developed a fourplex droplet digital PCR assay for PVL of BLV and susceptible and resistant host genes named IPATS-BLV. IPATS-BLV has inherent merits in measuring PVL and identifying susceptible and resistant cattle with superior simplicity and speed because of a single-well assay. Our new laboratory technique contributes to strengthening risk-based herd management used to control within-herd BLV transmission. Furthermore, this assay design potentially improves the diagnostics of other infectious diseases by combining the pathogen level and disease-related host genetic biomarker to predict disease outcomes.

Phylogenetic analysis based on whole genome sequence of bovine leukemia virus in cattle under 3 years old with enzootic bovine leukosis.

Enzootic bovine leukosis (EBL) is one of bovine neoplasms caused by bovine leukemia virus (BLV). Although EBL is typically observed in cattle over 3 years old, several cases of EBL onset in cattle under 3 years old have been reported in Japan. The mechanism for EBL onset in young cattle remains unclear. Although genetic variation of BLV is limited, the variations could affect viral properties relating to BLV pathogenesis. The purpose of this study was to clarify relationship between early onset of EBL and BLV groups. Moreover, we also aimed to characterize BLV that cause early onset of EBL. Whole genome sequences of BLV in 72 EBL cattle under 3 years old and 50 EBL cattle over 3 years old were identified. Phylogenetic analysis showed that BLV was divided into 4 groups (A, B-1, B-2 and Other). The BLV from EBL cattle under 3 years old were mainly classified as group A and B-1, while those from EBL cattle over 3 years old were mainly included in group B-2. Common sequence of group A and B-1 was compared with those of group B-2. Specific sequences in LTRs, gag-pro-pol, env and tax gene regions were identified in these groups. Amino acid substitutions of Pro and Tax protein were predicted in those nucleotide sequences. Those genetic variations might contribute to the early onset of EBL.

Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.

Detection of bovine leukemia virus in beef cattle kept in the Central Coast Regions of Vietnam.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leucosis. Our previous study showed the BLV existence in cattle kept in the Red River Delta Region of Vietnam. However, no positive samples were identified in beef cattle. Besides, information related to the BLV circulation in the remained parts of Vietnam is limited. Therefore, we tested the existence of BLV in 48 beef cattle kept in the Central Coast Regions. Nested PCR targeting the BLV-env-gp51 confirmed the prevalence of 14.6% in investigated regions. Phylogenetic analysis suggested the co-existence of genotypes 1 and 10. The close relationship between strains found in Vietnam, Thailand, Myanmar, and China was revealed suggesting the possibility of BLV transmission through the movement of live cattle.

Antigenicity of subregions of recombinant bovine leukemia virus (BLV) glycoprotein gp51 for antibody detection.

Bovine leukemia virus (BLV) is an enveloped virus, found worldwide that can infect cattle and induce many subclinical symptoms and malignant tumors. BLV infection causes severe economic losses in the cattle industry. The identification of BLV-infected cattle for segregation or elimination would be the most effective way to halt the spread of BLV infection on farms, owing to the lack of effective treatments and vaccines. Therefore, antibody detection against the viral glycoprotein gp51 is an effective method for diagnosing BLV-infected animals. In this study, ten different subregions of gp51 containing a common B cell epitope are vital for developing antigens as epitope-driven vaccine design and immunological assays. Such antigens were produced in Escherichia coli expression system to react with antibodies in the serum from BLV-infected cattle and compete for antigenicity. Recombinant His-gp5156-110 and gp5133-301(full) had the same sensitivity in BLV-positive sera, indicating that antibodies responded to the limited subregion of viral gp51, a common B cell epitope. This finding provides significant information for antigen selection in BLV to use in antibody detection assays. Further studies are needed to evaluate the antigenicity of His-gp5156-110 and gp5133-301(full) as antigens for antibody detection assays using a larger number of bovine serum samples.

Visualization of clonal expansion after massive depletion of cells carrying the bovine leukemia virus (BLV) integration sites during the course of disease progression in a BLV naturally-infected cow: a case report.

Bovine leukemia virus (BLV) infects cattle, integrates into host DNA as a provirus, and induces malignant B-cell lymphoma. Previous studies have addressed the impact of proviral integration of BLV on BLV-induced leukemogenesis. However, no studies have monitored sequential changes in integration sites in which naturally infected BLV individuals progress from the premalignant stage to the terminal disease. Here, we collected blood samples from a single, naturally infected Holstein cow at three disease progression stages (Stage I: polyclonal stage, Stage II: polyclonal toward oligoclonal stage, Stage III: oligoclonal stage) and successfully visualized the kinetics of clonal expansion of cells carrying BLV integration sites using our BLV proviral DNA-capture sequencing method. Although 24 integration sites were detected in Stages I and II, 92% of these sites experienced massive depletion in Stage III. Of these sites, 46%, 37%, and 17% were located within introns of Refseq genes, intergenic regions, and repetitive sequences, respectively. At Stage III cattle with lymphoma, only two integration sites were generated de novo in the intergenic region of Chr1, and the intron of the CHEK2 gene on Chr17 was significantly increased. Our results are the first to demonstrate clonal expansion after the massive depletion of cells carrying BLV integration sites in a naturally infected cow.