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Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc-allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution.

Background 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. Results 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. Conclusions 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.

Enzootic Bovine Leukosis (EBL) caused by the bovine leukemia virus (BLV) has been eradicated in over 20 countries. In contrast, the U.S. and many other nations are experiencing increasing prevalence in the absence of efforts to control transmission. Recent studies have shown that BLV infection in dairy cattle has a greater impact beyond the long-recognized lymphoma development that occurs in <5% of infected cattle. Like other retroviruses, BLV appears to cause multiple immune system disruptions, affecting both cellular and humoral immunity, which are likely responsible for increasingly documented associations with decreased dairy production and decreased productive lifespan. Realization of these economic losses has increased interest in controlling BLV using technology that was unavailable decades ago, when many nations eradicated BLV via traditional antibody testing and slaughter methods. This traditional control is not economically feasible for many nations where the average herd antibody prevalence is rapidly approaching 50%. The ELISA screening of cattle with follow-up testing via qPCR for proviral load helps prioritize the most infectious cattle for segregation or culling. The efficacy of this approach has been demonstrated in at least four herds. Breeding cattle for resistance to BLV disease progression also appears to hold promise, and several laboratories are working on BLV vaccines. There are many research priorities for a wide variety of disciplines, especially including the need to investigate the reports linking BLV and human breast cancer.

The Wnt signaling pathway plays key roles in differentiation and development and alterations in this signaling pathway are causally associated with numerous human diseases. While several laboratories were examining roles for Wnt signaling in skeletal development during the 1990s, interest in the pathway rose exponentially when three key papers were published in 2001-2002. One report found that loss of the Wnt co-receptor, Low-density lipoprotein related protein-5 (LRP5), was the underlying genetic cause of the syndrome Osteoporosis pseudoglioma (OPPG). OPPG is characterized by early-onset osteoporosis causing increased susceptibility to debilitating fractures. Shortly thereafter, two groups reported that individuals carrying a specific point mutation in LRP5 (G171V) develop high-bone mass. Subsequent to this, the causative mechanisms for these observations heightened the need to understand the mechanisms by which Wnt signaling controlled bone development and homeostasis and encouraged significant investment from biotechnology and pharmaceutical companies to develop methods to activate Wnt signaling to increase bone mass to treat osteoporosis and other bone disease. In this review, we will briefly summarize the cellular mechanisms underlying Wnt signaling and discuss the observations related to OPPG and the high-bone mass disorders that heightened the appreciation of the role of Wnt signaling in normal bone development and homeostasis. We will then present a comprehensive overview of the core components of the pathway with an emphasis on the phenotypes associated with mice carrying genetically engineered mutations in these genes and clinical observations that further link alterations in the pathway to changes in human bone.

About the Authors: Cassandra R. Diegel Affiliation: Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America Steven Hann Affiliation: Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America Ugur M. Ayturk Affiliations Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, Musculoskeletal Integrity Program, Hospital for Special Surgery Research Institute, New York, New York, United States of America Jennifer C. W. Hu Affiliation: Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America Kyung-Eun Lim Affiliation: Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America Casey J. Droscha Affiliation: Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America Zachary B. Madaj Affiliation: Bioinformatics and Biostatistics Core, Van Andel Institute, Grand Rapids, Michigan, United States of America Gabrielle E. Foxa Affiliation: Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America Isaac Izaguirre Affiliation: Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America VAI Vivarium and Transgenics Core ¶Members of the VAI Vivarium and Transgenics Core are listed in the Acknowledgments Affiliation: Vivarium and Transgenics Core, Van Andel Institute, Grand Rapids, Michigan, United States of America Alexander G. Robling Affiliation: Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America Matthew L. Warman Affiliation: Orthopedic Research Labs, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America Bart O. Williams * E-mail: bart.williams@vai.org Affiliation: Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America ORCID logo http://orcid.org/0000-0002-5261-5301 Citation: Diegel CR, Hann S, Ayturk UM, Hu JCW, Lim K-E, Droscha CJ, et al. Furthermore, the osteocalcin-null rat model did not develop obesity, insulin resistance, or glucose intolerance, which conflicts with Dr. Karsenty’s mice [6]. Committee on Responsible Science, Committee on Science Engineering Medicine and Public Policy (U.S.) (2017) Fostering integrity in research. 1 online resource (1 PDF file (xv, 307 pages)) p.

Abstract Background There is currently no commercially available method in Canada to identify bovine leukemia virus (BLV)-positive cows with high proviral load (PVL). Objectives First, develop a model to predict PVL using common, commercially available, cost-effective diagnostic tests. Second, investigate the relationship between lymphocyte count and PVL in BLV-positive cows. Animals A total of 339 BLV-positive and 62 BLV-seronegative cows on 15 dairy farms. Methods Cross-sectional study. Blood and milk samples were collected from all lactating BLV-positive cows on each farm and 5 to 10 BLV-seronegative cows depending on herd size. Blood and milk samples were tested for anti-BLV antibodies using enzyme-linked immunosorbent assay (ELISA). Complete blood counts were performed on blood samples, and standard components analyses were obtained for milk samples. Proviral load was determined by quantitative polymerase chain reaction for each cow. Results The inverse of lymphocyte count, the square of the inverse of lymphocyte count, and milk ELISA percent positivity were positively associated with increasing PVL in BLV-positive cows. For BLV-positive cows, lymphocyte count >5.2 × 109/L predicted a high PVL (BLV:Bovine DNA of >1 in blood) with a sensitivity of 92.4% and a specificity of 79.8%. For BLV-positive cows, white blood cell count >10.8 × 109/L predicted a high PVL, with a sensitivity of 85.5% and a specificity of 83.6%. Conclusions and Clinical Importance Based on these results, producers can implement commonly available diagnostic tests to identify cows with high probability of having high PVL, which may help in designing effective disease control strategies for BLV-positive herds.

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.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease in cattle. Previous work estimates that 78% of US beef operations and 38% of US beef cattle are seropositive for BLV. Infection by BLV in a herd is an economic concern for producers as evidence suggests that it causes an increase in cost and a subsequent decrease in profit to producers. Studies investigating BLV in dairy cattle have noted disease resistance or susceptibility, measured by a proviral load (PVL) associated with specific alleles of the bovine leukocyte antigen (BoLA) DRB3 gene. This study aims to investigate the associations between BoLA DRB3 alleles and BLV PVL in beef cattle. Samples were collected from 157 Midwest beef cows. BoLA DRB3 alleles were identified and compared with BLV PVL. One BoLA DRB3 allele, *026:01, was found to be associated with high PVL in relation to the average of the sampled population. In contrast, two alleles, *033:01 and *002:01, were found to be associated with low PVL. This study provides evidence of a relationship between BoLA DRB3 alleles and BLV PVL in US beef cows.

Bovine leukemia virus (BLV) is estimated to infect over 83% of dairy herds and over 40% of all dairy cows in the United States. While, BLV only causes leukemia in a small proportion of animals, research indicates that BLV+ cattle exhibit reduced milk production and longevity that is distinct from lymphoma development. It is hypothesized that BLV negatively affects production by interfering with cattle immunity and increasing the risk of secondary infections. In particular, BLV+ cows demonstrate reduced circulating levels of both antigen-specific and total IgM. This study investigated possible mechanisms by which BLV could interfere with the production of IgM in naturally infected cattle. Specifically, total plasma IgM and the expression of genes , and in circulating IgM+ B cells were measured in 15 naturally infected BLV+ and 15 BLV- cows. In addition, BLV proviral load (PVL) (a relative measurement of BLV provirus integrated into host DNA) and the relative expression of BLV and 5 BLV microRNAs (miRNAs) were characterized and correlated to the expression of selected endogenous genes. BLV+ cows exhibited lower total plasma IgM and lower expression of , and . While, BLV and BLV miRNAs failed to correlate with expression, both BLV and BLV miRNAs exhibited negative associations with and gene expression. The results suggest a possible transcriptional pathway by which BLV interferes with IgM production in naturally infected cattle.

Characterization of the bovine leukocyte antigen (BoLA) DRB3 gene has shown that specific alleles associate with susceptibility or resilience to the progression of bovine leukemia virus (BLV), measured by proviral load (PVL). Through surveillance of multi-farm BLV eradication field trials, we observed differential phenotypes within seropositive cows that persist from months to years. We sought to develop a multiplex next-generation sequencing workflow (NGS-SBT) capable of genotyping 384 samples per run to assess the relationship between BLV phenotype and two BoLA genes. We utilized longitudinal results from milk ELISA screening and subsequent blood collections on seropositive cows for PVL determination using a novel BLV proviral load multiplex qPCR assay to phenotype the cows. Repeated diagnostic observations defined two distinct phenotypes in our study population, ELISA-positive cows that do not harbor detectable levels of provirus and those who do have persistent proviral loads. In total, 565 cows from nine Midwest dairy farms were selected for NGS-SBT, with 558 cows: 168 BLV susceptible (ELISA-positive/PVL-positive) and 390 BLV resilient (ELISA-positive/PVL-negative) successfully genotyped. Three BoLA-DRB3 alleles, including one novel allele, were shown to associate with disease resilience, *009:02, *044:01, and *048:02 were found at rates of 97.5%, 86.5%, and 90.3%, respectively, within the phenotypically resilient population. Alternatively, DRB3*015:01 and *027:03, both known to associate with disease progression, were found at rates of 81.1% and 92.3%, respectively, within the susceptible population. This study helps solidify the immunogenetic relationship between BoLA-DRB3 alleles and BLV infection status of these two phenotypic groupings of US dairy cattle.