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.

Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus.

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

Prevalence and spatial distribution of infectious diseases of dairy cattle in Ontario, Canada.

Here we investigated the prevalence and spatial distribution of selected pathogens associated with infectious diseases of dairy cattle in Ontario, Canada. The cross-sectional study surveyed bulk tank milk for antibodies against bovine leukemia virus (BLV), Mycobacterium avium ssp. paratuberculosis (MAP), and Salmonella Dublin, and for the presence of mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis). Between October 2021 and June 2022, bulk tank milk (BTM) samples were obtained from every commercial dairy farm in Ontario (n = 3,286). Samples underwent ELISA testing for presence of BLV, MAP and S. Dublin antibodies, and quantitative PCR testing for the detection of specific antigens of pathogens associated with mastitis. Bayesian models were used to estimate prevalence, and spatial analysis was carried out to identify regional clusters of high pathogen prevalence. Prevalence varied for different pathogens. BLV was widespread across dairy farms in Ontario, with an estimated prevalence of 88.3%. Prevalence of MAP, Staph. aureus and S. Dublin in Ontario dairy herds were 39.8%, 31.5% and 5.1%, respectively. The vast majority of dairy herds in Ontario were free of intramammary infections caused by Strep. agalactiae and M. bovis. Clusters of increased test positivity rates were detected for S. Dublin, MAP, and Staph. aureus, indicating potential geographic risk factors for pathogen transmission. For S. Dublin, an area of increased test positivity rates was detected in southwestern Ontario, close to the Canada-US border where most of the dairy herds in Ontario are located. Conversely, a localized cluster of positive test outcomes involving 14 subdivisions located in the southeastern region of Ontario was detected for Staph. aureus. Findings from our survey highlight the importance of the testing of aggregated samples and spatial analysis as part of disease surveillance programs and for implementing risk-based trading approaches among dairy producers.

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.

The Global Epidemiology of Bovine Leukemia Virus: Current Trends and Future Implications.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis (EBL), which is the most significant neoplastic disease in cattle. Although EBL has been successfully eradicated in most European countries, infections continue to rise in Argentina, Brazil, Canada, Japan, and the United States. BLV imposes a substantial economic burden on the cattle industry, particularly in dairy farming, as it leads to a decline in animal production performance and increases the risk of disease. Moreover, trade restrictions on diseased animals and products between countries and regions further exacerbate the problem. Recent studies have also identified fragments of BLV nucleic acid in human breast cancer tissues, raising concerns for public health. Due to the absence of an effective vaccine, controlling the disease is challenging. Therefore, it is crucial to accurately detect and diagnose BLV at an early stage to control its spread and minimize economic losses. This review provides a comprehensive examination of BLV, encompassing its genomic structure, epidemiology, modes of transmission, clinical symptoms, detection methods, hazards, and control strategies. The aim is to provide strategic information for future BLV research.

The influence of risk factors on bovine leukemia virus infection and proviral load in egyptian cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), which affects cattle globally. In Egypt, BLV control strategies have been ignored because of the shortage of BLV research studies and the silent infection in most animals. This study aimed to identify the risk factors associated with the prevalence of BLV among dairy and beef cattle from six different geographic and climatic provinces in Egypt. Additionally, risk factors affecting the BLV proviral load (PVL) among the positive cattle were targeted. The total BLV prevalence in cattle from six investigated Egyptian provinces was 24.2% (105/433), while the mean PVL (8651.6 copies /105 white blood cells) was absolutely high as estimated by the BLV-CoCoMo-quantitative polymerase chain reaction (qPCR)-2 assay. Analysis of the influence of risk factors (age, sex, breed, production type, farm size, and location) on BLV prevalence indicated that the Holstein breed (OR = 1.582, p = 0.007), beef cattle (OR = 1.088, p = 0.0001), large-size farms (OR = 1.26, p = 0.0001), and cattle from Damietta (OR = 1.43, p = 0.0001) and Cairo (OR = 1.16, p = 0.0001) were ultimately proven the most important risks for BLV infection. The risk factors were analyzed considering the BLV PVL levels in the BLV-positive cases. Significantly high PVL (HPVL) levels were observed in cattle > 5 years old (p < 0.0001), females (p = 0.0008), Holstein (p < 0.0001), dairy cows (p = 0.0053), large-size farms (p < 0.0001), and cattle from Damietta (p < 0.0001) compared to other categories. Contrary, no significant differences in PVL levels were reported between the Native and Mixed cattle breeds (p = 0.13). Ultimately, the logistic regression model indicated that the probability of carrying HPVL in cattle > 5 years is 1.27 (95% CI: 1.03-2.09, p < 0.001) times more likely compared to cattle < 2 years old. In conclusion, the findings were valuably correlating the BLV prevalence with PVL as an indicator of the risk of BLV infection.

Estimation of the proviral load in Japanese Black cattle infected with bovine leukemia virus by statistical modeling.

Enzootic bovine leukosis (EBL) is B-cell lymphoma in cattle caused by bovine leukemia virus (BLV) infection. The incidence of EBL has been increasing since 1998 in Japan, resulting in significant economic losses for farms. The BLV genome integrates with the host genome as provirus, leading to sustainably infection. Although most of the BLV-infected cattle are aleukemic, some cattle cause persistent lymphocytosis (PL) and subsequently develop EBL. Recent reports suggest the association between the risk for the transmission of BLV and the developing EBL and the proviral load (PVL) in BLV-infected cattle, which cannot measure readily in the field. This study aims to build a statistical model for predicting PVL of BLV-infected asymptomatic or PL cattle based on data accessible in the field. Five negative binomial regression models with different linear predictors were built and compared for the predictability of PVL. Consequently, the model with two explanatory variables (age in months and logarithm of lymphocyte count) was selected as the best model. The model can be used in the field as a cost-beneficial supporting tool to estimate the risk of transmission of BLV and developing EBL in infected cattle.

Predicting an increased risk of severe clinical mastitis and economic loss using a threshold value of bovine leukemia virus proviral load.

OBJECTIVE: To establish a threshold value of bovine leukemia virus (BLV) proviral load (PVL) to identify increased risk of severe clinical mastitis, and to examine the prognosis and economic loss of clinical mastitis based on the newly established PVL cut-off value. ANIMALS: 97 lactating Holstein cows with clinical mastitis. METHODS: Blood and milk samples were collected aseptically from each cow. Youden index was used for receiver-operating characteristic curve analysis with the severity rate of clinical mastitis as the dependent variable and PVL as an independent variable. PVL cut-off value was used as a criterion to compare the severity rate of clinical mastitis, percentage of cows with and without systemic treatments, number of treatments, cost of treatment, and prognosis. RESULTS: PVL cut-off value was 17.8 copies/10 ng DNA for the dependent variable MILD vs SEVERE. The severity rate of clinical mastitis, percentage of cows given systemic treatments, and technical fees for medical treatment were significantly higher in the group above the PVL cut-off value than in the group below the PVL cut-off value and the negative group. Number of treatments was significantly higher in the group above the cut-off value than in the group below the cut-off value. There was no significant difference in prognosis after mastitis among the 3 groups. CLINICAL RELEVANCE: These results suggested that PVL cut-off value of 17.8 copies/10 ng DNA was a useful threshold for increased economic losses in BLV-infected cows; it may also serve as a new standard value for the detection and culling of BLV-infected cows in Japan.

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.

Influence of Maternal BLV Infection on miRNA and tRF Expression in Calves.

Small non-coding RNAs, such as microRNAs (miRNA) and tRNA-derived fragments (tRF), are known to be involved in post-transcriptional gene regulation. Research has provided evidence that small RNAs may influence immune development in calves. Bovine leukosis is a disease in cattle caused by Bovine Leukemia Virus (BLV) that leads to increased susceptibility to opportunistic pathogens. No research has addressed the potential influence that a maternal BLV infection may have on gene regulation through the differential expression of miRNAs or tRFs in progeny. Blood samples from 14-day old Holstein calves born to BLV-infected dams were collected. Antibodies for BLV were assessed using ELISA and levels of BLV provirus were assessed using qPCR. Total RNA was extracted from whole blood samples for small RNA sequencing. Five miRNAs (bta-miR-1, bta-miR-206, bta-miR-133a, bta-miR-133b, and bta-miR-2450d) and five tRFs (tRF-36-8JZ8RN58X2NF79E, tRF-20-0PF05B2I, tRF-27-W4R951KHZKK, tRF-22-S3M8309NF, and tRF-26-M87SFR2W9J0) were dysregulated in calves born to BLV-infected dams. The miRNAs appear to be involved in the gene regulation of immunological responses and muscle development. The tRF subtypes and parental tRNA profiles in calves born to infected dams appear to be consistent with previous publications in adult cattle with BLV infection. These findings offer insight into how maternal BLV infection status may impact the development of offspring.

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.

Characterization of mRNA Signature in Milk Small Extracellular Vesicles from Cattle Infected with Bovine Leukemia Virus.

This study aimed to characterize the mRNA signature of milk small extracellular vesicles (sEVs) from BLV-infected cattle. A total of 23 mRNAs, which showed greater abundance in milk sEVs from BLV-infected cattle compared to those from BLV-uninfected (control) cattle, were identified through microarray analyses conducted in our previous study. To assess the significance of these differences in mRNA abundance, milk was collected from six control cattle and twenty-six cattle infected with BLV. The infected cattle were categorized into two distinct groups based on their proviral loads: a group of eight cattle with low proviral loads (LPVL), characterized by <10,000 copies per 105 white blood cells (WBC), and a group of eighteen cattle with high proviral loads (HPVL), marked by ≥10,000 copies per 105 WBC. The qPCR analysis quantified 7 out of 23 mRNAs, including BoLA, CALB1, IL33, ITGB2, MYOF, TGFBR1, and TMEM156, in the milk sEVs from control cattle, LPVL cattle, and HPVL cattle. Significantly, the average relative expression of CALB1 mRNA in milk sEVs was higher in LPVL cattle compared to HPVL cattle and control cattle (p < 0.05), while it was relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). Likewise, the average relative expression of TMEM156 mRNA in milk sEVs was significantly higher in LPVL cattle compared to HPVL cattle (p < 0.05), and relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). The results indicate distinct patterns of CALB1 and TMEM156 mRNA levels in milk sEVs, with higher levels observed in LPVL cattle and lower levels in HPVL cattle. The current study could provide essential information to comprehend the complexities during the progression of BLV infection and direct the exploration of mRNA biomarkers for monitoring the clinical stage of BLV infection.

Oncogenic viral DNA related to human breast cancer found on cattle milk and meat.

Bovine leukemia virus (BLV) is a major cause of lymphoma in cattle and has been recently correlated to breast cancer in humans. How and whether BLV might reach humans remains unknown but it could be through cattle-derived milk and meat. Here our aim was to investigate whether BLV DNA could be found in fresh milk and raw meat destined to human consumption and whether anti-BLV antibodies could be detected in human blood at the same geographical region. Milk (n = 36) and meat (n = 54) samples were collected from cows knowingly seropositive or negative to BLV and evaluated by nested PCR targeting BLV tax gene. Human serum samples (n = 900) were tested by ELISA to detect anti-BLV antibodies. BLV DNA was detected in 39 % of the milk samples and in 32 % of meat samples from BLV positive cows. Anti-BLV antibodies were found in 4.1 % of the human serum samples. Our data further supports the hypothesis that BLV might cause a zoonotic infection and indicate that milk and meat from BLV-infected cattle might be considered a potential source of infection to humans.

Seroprevalence and detection of bovine leukosis virus in semen from breeding bulls in Costa Rica.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a persistent infection and the most important neoplastic disease in cattle. It is spread primarily by transferring infected lymphocytes through blood from carriers to healthy animals. The present study is aimed at determining the seropositivity of BLV in breeding bulls from Costa Rica and at detecting for the first time in the country BLV DNA in bull semen. Between May 2011 and August 2018, 379 blood and 133 semen samples were collected from bulls distributed in 118 farms. The serum was analyzed by an enzymatic immunoassay and the semen by polymerase chain reaction and sequencing. BLV seropositivity was 43.5% (165/379), while 64.4% (76/118) of the farms had positive reactors. Holstein (75.7%) and Jersey (73.0%) breeds showed the highest seropositivity. In addition, Bos taurus bulls (68.1%), older than seven years (50.0%), and those belonging to dairy farms (75.5%) had higher seropositivity compared to Bos indicus (17.7%), younger than seven years (42.2%), and those from beef farms (15.5%), respectively. Moreover, Bos taurus bulls had a higher risk of being seropositive than Bos indicus (OR = 3.4; 95% CI: 1.7-6.8). BLV DNA was found in one semen sample (2.5%; 1/40) from a seropositive bull. The importance of serum and molecular BLV screening in semen samples and the potential role of some risk factors associated with the disease, such as the bull's age, genotype, and type of livestock productive system, is argued in the present report.

The bovine leukemia virus-derived long non-coding RNA AS1-S binds to bovine hnRNPM and alters the interaction between hnRNPM and host mRNAs.

Viruses utilize several strategies to cause latent infection and evade host immune responses. Long non-coding RNA (lncRNA), a class of non-protein-encoding RNA that regulates various cellular functions by interacting with RNA-binding proteins, plays important roles for viral latency in several viruses, such as herpesviruses and retroviruses, due to its lack of antigenicity. Bovine leukemia virus (BLV), which belongs to the family Retroviridae, encodes the BLV-derived lncRNA AS1-S, which is a major transcript expressed in latently infected cells. We herein identified bovine heterogeneous nuclear ribonucleoprotein M (hnRNPM), an RNA-binding protein located in the nucleus, as the binding partner of AS1-S using an RNA-protein pull-down assay. The pull-down assay using recombinant hnRNPM mutants showed that RNA recognition motifs (RRMs) 1 and 2, located in the N-terminal region of bovine hnRNPM, were responsible for the binding to AS1-S. Furthermore, RNA immunoprecipitation (RIP) assay results showed that the expression of AS1-S increased the number of mRNAs that co-immunoprecipitated with bovine hnRNPM in MDBK cells. These results suggested that AS1-S could alter the interaction between hnRNPM and host mRNAs, potentially interfering with cellular functions during the initial phase of mRNA maturation in the nucleus. Since most of the identified mRNAs that exhibited increased binding to hnRNPM were correlated with the KEGG term "Pathways in cancer," AS1-S might affect the proliferation and expansion of BLV-infected cells and contribute to tumor progression. IMPORTANCE BLV infects bovine B cells and causes malignant lymphoma, a disease that greatly affects the livestock industry. Due to its low incidence and long latent period, the molecular mechanisms underlying the progression of lymphoma remain enigmatic. Several non-coding RNAs (ncRNAs), such as miRNA and lncRNA, have recently been discovered in the BLV genome, and the relationship between BLV pathogenesis and these ncRNAs is attracting attention. However, most of the molecular functions of these transcripts remain unidentified. To the best of our knowledge, this is the first report describing a molecular function for the BLV-derived lncRNA AS1-S. The findings reported herein reveal a novel mechanism underlying BLV pathogenesis that could provide important insights for not only BLV research but also comparative studies of retroviruses.

Exploration of microRNA Biomarkers in Blood Small Extracellular Vesicles for Enzootic Bovine Leukosis.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). While most infected cattle show no clinical signs, approximately 30% of infected cattle develop persistent lymphocytosis (PL), and a small percentage may develop EBL. Currently, there is no method for predicting the possibility of EBL onset. In this study, we analyzed the microRNAs (miRNAs) encapsulated in small extracellular vesicles (sEVs) in the blood to explore the biomarkers of EBL. To identify candidate biomarkers, blood samples were collected from three BLV-uninfected and three EBL cattle. Total RNA was extracted from filtered serum and used for microarray analysis. Due to their association with cancer in human orthologs, we selected three miRNAs as candidate biomarkers, bta-miR-17-5p, bta-miR-24-3p, and bta-miR-210, which were more than twice as abundant in EBL cattle than in BLV-uninfected cattle. Quantitative real-time polymerase chain reaction (qPCR) using serum RNAs from six cattle used for the microarray analysis was carried out for the detection of the three selected miRNAs. Additionally, bta-miR-92a, whose ortholog has been associated with cancer in humans, was also examined by qPCR. bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were successfully detected, but bta-miR-210 was not. To further evaluate the utility of these three miRNAs as biomarkers, new blood samples were collected from 31 BLV-uninfected and 30 EBL cattle. The levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were significantly higher in EBL cattle than in BLV-uninfected cattle. These results suggest that increased levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a in the blood could be used as biomarkers for EBL. This study may contribute to the control of BLV infections and develop a prediction method of EBL onset.

Effectiveness of integrated bovine leukemia virus eradication strategies utilizing cattle carrying resistant and susceptible major histocompatibility complex class II DRB3 alleles.

Bovine leukemia virus (BLV) has spread worldwide and causes serious problems in the cattle industry owing to the lack of effective treatments and vaccines. Bovine leukemia virus is transmitted via horizontal and vertical infection, and cattle with high BLV proviral load (PVL), which is a useful index for estimating disease progression and transmission risk, are considered major infectious sources within herds. The PVL strongly correlates with highly polymorphic bovine lymphocyte antigen (BoLA)-DRB3 alleles. The BoLA-DRB3*015:01 and *012:01 alleles are known susceptibility-associated markers related to high PVL, and cattle with susceptible alleles may be at a high risk of BLV transmission via direct contact with healthy cows. In contrast, the BoLA-DRB3*009:02 and *014:01:01 alleles comprise resistant markers associated with the development of low PVL, and cattle with resistant alleles may be low-risk spreaders for BLV transmission and disrupt the BLV transmission chain. However, whether polymorphisms in BoLA-DRB3 are useful for BLV eradication in farms remains unknown. Here, we conducted a validation trial of the integrated BLV eradication strategy to prevent new infection by resistant cattle and actively eliminate susceptible cattle in addition to conventional BLV eradication strategies to maximally reduce the BLV prevalence and PVL using a total of 342 cattle at 4 stall-barn farms in Japan from 2017 to 2019. First, we placed the resistant milking cattle between the BLV-positive and BLV-negative milking cattle in a stall barn for 3 yr. Interestingly, the resistant cattle proved to be an effective biological barrier to successfully block the new BLV infections in the stall-barn system among all 4 farms. Concomitantly, we actively eliminated cattle with high PVL, especially susceptible cattle. Indeed, 39 of the 60 susceptible cattle (65%), 76 of the 140 neutral cattle (54%), and 20 of the 41 resistant cattle (48.8%) were culled on 4 farms for 3 years. Consequently, BLV prevalence and mean PVL decreased in all 4 farms. In particular, one farm achieved BLV-free status in May 2020. By decreasing the number of BLV-positive animals, the revenue-enhancing effect was estimated to be ¥5,839,262 ($39,292.39) for the 4 farms over 3 yr. Our results suggest that an integrated BLV eradication program utilization of resistant cattle as a biological barrier and the preferential elimination of susceptible cattle are useful for BLV infection control.

Bovine leukemia virus detection in humans: A systematic review and meta-analysis.

To review the available studies on the frequency of detection of the bovine leukemia virus in human samples, a systematic review with meta-analysis of the scientific literature was carried out, including papers published in English, Spanish, and Portuguese in 5 multidisciplinary databases. We collected information from different populations following a detailed and reproducible search protocol in which two researchers verified the inclusion and exclusion criteria. We identified 759 articles, of which only 33 met the inclusion criteria. Analyzed studies reported that the presence of the virus was measured in human samples, such as paraffin-embedded breast tissue and peripheral blood from 10,398 individuals, through serological and molecular techniques. An overall virus frequency of 27% (Ranging between 17 and 37%) was observed, with a high-frequency data heterogeneity between studies. The presence of this virus in different human biological samples suggests the need to investigate further its transmission route to humans and its potential role in developing and progressing diseases.

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.

Development of a novel fluorogenic assay method for screening inhibitors of bovine leukemia virus protease and identification of mitorubrinic acid as an anti-BLV compound.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, a fatal cattle disease that leads to significant economic losses in the livestock industry. Currently, no effective BLV countermeasures exist, except testing and culling. In this study, we developed a high-throughput fluorogenic assay to evaluate the inhibitory activity of various compounds on BLV protease, an essential enzyme for viral replication. The developed assay method was used to screen a chemical library, and mitorubrinic acid was identified as a BLV protease inhibitor that exhibited stronger inhibitory activity than amprenavir. Additionally, the anti-BLV activity of both compounds was evaluated using a cell-based assay, and mitorubrinic acid was found to exhibit inhibitory activity without cytotoxicity. This study presents the first report of a natural inhibitor of BLV protease-mitorubrinic acid-a potential candidate for the development of anti-BLV drugs. The developed method can be used for high-throughput screening of large-scale chemical libraries.