Comprehensive Comparison of Novel Bovine Leukemia Virus (BLV) Integration Sites between B-Cell Lymphoma Lines BLSC-KU1 and BLSC-KU17 Using the Viral DNA Capture High-Throughput Sequencing Method

Bovine leukemia virus (BLV) infects cattle and integrates into host DNA, causing enzootic bovine leukosis (EBL), an aggressive B-cell lymphoma. Here, we developed a novel proviral DNA-capture sequencing (proviral DNA-capture-seq) method investigating BLV proviral integration in two B-cell lymphoma lines, BLSC-KU1 and BLSC-KU17, derived from BLV-infected cattle with EBL. We designed BLV-specific biotinylated probes to capture the provirus genome and enrich libraries for next-generation sequencing. Validation showed high specificity and efficient enrichment of target sequence reads as well as identification of three BLV proviral integration sites on BLV persistently infected FLK-BLV cells as a positive control. We successfully detected a single BLV proviral integration site on chromosome 19 of BLSC-KU1 and chromosome 9 of BLSC-KU17, which were confirmed by standard PCR and Sanger sequencing. Further, a defective provirus in BLSC-KU1 and complete BLV proviral sequence in BLSC-KU17 were confirmed using long PCR and sequencing. This is the first study to provide comprehensive information on BLV proviral structure and viral integration in BLSC-KU1 and BLSC-KU17. Moreover, the proposed method can facilitate understanding of the detailed mechanisms underlying BLV-induced leukemogenesis and may be used as an innovative tool to screen BLV-infected cattle at risk at an earlier stage than those that have already developed lymphoma.     

Bovine Leukaemia Virus: Current Epidemiological Circumstance and Future Prospective

Bovine leukaemia virus (BLV) is a deltaretrovirus that is closely related to human T-cell leukaemia virus types 1 and 2 (HTLV-1 and -2). It causes enzootic bovine leukosis (EBL), which is the most important neoplastic disease in cattle. Most BLV-infected cattle are asymptomatic, which potentiates extremely high shedding rates of the virus in many cattle populations. Approximately 30% of them show persistent lymphocytosis that has various clinical outcomes; only a small proportion of animals (less than 5%) exhibit signs of EBL. BLV causes major economic losses in the cattle industry, especially in dairy farms. Direct costs are due to a decrease in animal productivity and in cow longevity; indirect costs are caused by restrictions that are placed on the import of animals and animal products from infected areas. Most European regions have implemented an efficient eradication programme, yet BLV prevalence remains high worldwide. Control of the disease is not feasible because there is no effective vaccine against it. Therefore, detection and early diagnosis of the disease are essential in order to diminish its spreading and the economic losses it causes. This review comprises an overview of bovine leukosis, which highlights the epidemiology of the disease, diagnostic tests that are used and effective control strategies.     

Comparative Evaluation of Three Commercial Quantitative Real-Time PCRs Used in Japan for Bovine Leukemia Virus

Bovine leukemia virus (BLV) is an oncogenic virus belonging to the genus Deltaretrovirus and is the causative agent of enzootic bovine leukosis. Proviral load (PVL) determined by real-time quantitative PCR (qPCR) is now widely used as an indicator of not only BLV infection, but also BLV disease progression. To interpret PVLs determined by different qPCRs used in Japan, we compared a chimeric cycling probe-based qPCR, CY415, targeting the BLV tax region; a TaqMan probe-based qPCR, RC202, targeting the BLV pol region; and a TaqMan probe-based qPCR, CoCoMo, targeting the BLV long terminal repeat (LTR) region. Whole-blood samples collected from 317 naturally BLV-infected cattle (165 Holstein–Friesian and 152 Japanese Black) and tumor tissue samples collected from 32 cattle at a meat inspection center were used. The PVLs determined by each qPCR were strongly correlated. However, the PVL and the proportion of BLV-infected cells determined by RC202 or CoCoMo were significantly higher than those determined by CY415. Genetic analysis of three tumor tissue samples revealed that LTR region mutations or a deletion affected the PVL determined by CoCoMo. These results suggest that the TaqMan-based RC202 or CoCoMo qPCR is better than CY415 for BLV PVL analysis. However, qPCR target region mutations were not rare in tumors and could hamper PVL analysis by using qPCR.     

Diversity and evolution of 11 innate immune genes in Bos taurus taurus and Bos taurus indicus cattle

The Toll-like receptor (TLR) and peptidoglycan recognition protein 1 (PGLYRP1) genes play key roles in the innate immune systems of mammals. While the TLRs recognize a variety of invading pathogens and induce innate immune responses, PGLYRP1 is directly microbicidal. We used custom allele-specific assays to genotype and validate 220 diallelic variants, including 54 nonsynonymous SNPs in 11 bovine innate immune genes (TLR1-TLR10, PGLYRP1) for 37 cattle breeds. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and we were unable to differentiate between the specialized B. t. taurus beef and dairy breeds, despite an average polymorphism density of one locus per 219 bp. Ninety-nine tagSNPs and one tag insertion-deletion polymorphism were sufficient to predict 100% of the variation at all 11 innate immune loci in both subspecies and their hybrids, whereas 58 tagSNPs captured 100% of the variation at 172 loci in B. t. taurus. PolyPhen and SIFT analyses of nonsynonymous SNPs encoding amino acid replacements indicated that the majority of these substitutions were benign, but up to 31% were expected to potentially impact protein function. Several diversity-based tests provided support for strong purifying selection acting on TLR10 in B. t. taurus cattle. These results will broadly impact efforts related to bovine translational genomics.     

Bacterial lipopolysaccharide-induced coordinate downregulation of arginine vasopressin receptor V3 and corticotropin-releasing factor receptor 1 messenger ribonucleic acids in the anterior pituitary of endotoxemic steers

AVP and CRF are potent stimulators of pituitary ACTH secretion in cattle. Actions of AVP and CRF at the anterior pituitary are mediated by AVP receptor V3 (V3) and CRF receptor 1 (CRFR1). The primary objective of these studies was to determine the effect of systemic inflammatory stress on V3 and CRFR1 mRNAs in the bovine anterior pituitary. Holstein steers (n=20) were injected with 200 ng/kg bacterial lipopolysaccharide (LPS) and tissues collected 0, 2, 4, 12, and 24 h later. All animals responded to LPS administration with an increase in body temperature, plasma ACTH, and cortisol (p<0.05). Abundance of anterior pituitary V3 mRNA was decreased at 2, 4, and 12 h following LPS administration (p<0.05) and returned to basal by 24 h. A similar temporal regulation of pituitary CRFR1 mRNA (p<0.05), but not pituitary pro-opiomelanocortin (POMC) mRNA, was observed following LPS administration. Similar downregulation of CRFR1 mRNA was not observed in other brain regions following LPS administration (cerebellum, hypothalamus). Our results indicate that V3 and CRFR1 mRNAs are coordinately downregulated in the anterior pituitary during systemic inflammatory stress. Decreased AVP and CRF receptor expression may help regulate the pituitary-adrenal response to stress.     

Gene expression analysis of embryonic pancreas development master regulators and terminal cell fate markers in resected pancreatic cancer: A correlation with clinical outcome

Background

Despite the recent introduction of new drugs and the development of innovative multi-target treatments, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains very poor. Even when PDAC is resectable, the rate of local or widespread disease recurrence remains particularly high. Currently, reliable prognostic biomarkers of recurrence are lacking. We decided to explore the potential usefulness of pancreatic developmental regulators as biomarkers of PDAC relapse.

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with ³²P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.     

Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10^?9 m melatonin. We analyzed the ROS, mitochondrial Ca²⁺ (mCa²⁺) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase‐3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa²⁺, Bax mRNA, and caspase‐3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa²⁺, Bax mRNA expression, and caspase‐3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10^?9 m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.     

Further studies on the antigenic properties and distribution of the putative bovine leukemia virus.

The C-type viruses found in long-term cultures. New Bolton Center (NBC) cell lines, of peripheral lymphocytes from leukemic cattle and in short-term cultures of bovine buffy coat(BC) cells share an immunofluorescent(IF)antigen detected in the cytoplasm of infected cells as well as an antigen demonstrable in gel diffusion experiments. Therefore the viruses from these cultures most likely represent different isolates of the putative bovine leukemia virus (BLV). The BLV precipitin antigen is analogous to the group specific (gs) antigens of the leukemia viruses of other species in that it is soluble, ether resistant, and apparently located within the virion. These observations, together with results showing that the specificity of the BLV precipitin antigen differs from that of the gs antigen of other mammalian leukemia viruses, indicate that the former antigen represents the intraspecies (gs-1) determinant of BLV. Antibodies to the precipitin viral antigen were found in 82% of cattle with leukemia and in 40% of clinically normal adult cattle in multiple-case herds. These groups of animals also had fluorescent antibodies to the virus, but with significantly higher frequencies (100% and 76%, respectively). On the other hand, in leukemia-free herds, precipitating antibodies were not found and the incidence of fluorescent antibodies was only 3%.     

A Novel Subtype of Bovine Hepacivirus Identified in Ticks Reveals the Genetic Diversity and Evolution of Bovine Hepacivirus

Hepaciviruses represent a group of viruses that pose a significant threat to the health of humans and animals. New members of the genus Hepacivirus in the family Flaviviridae have recently been identified in a wide variety of host species worldwide. Similar to the Hepatitis C virus (HCV), bovine hepacivirus (BovHepV) is hepatotropic and causes acute or persistent infections in cattle. BovHepVs are distributed worldwide and classified into two genotypes with seven subtypes in genotype 1. In this study, three BovHepV strains were identified in the samples of ticks sucking blood on cattle in the Guangdong province of China, through unbiased high-throughput sequencing. Genetic analysis revealed the polyprotein-coding gene of these viral sequences herein shared 67.7–84.8% nt identity and 76.1–95.6% aa identity with other BovHepVs identified worldwide. As per the demarcation criteria adopted for the genotyping and subtyping of HCV, these three BovHepV strains belonged to a novel subtype within the genotype 1. Additionally, purifying selection was the dominant evolutionary pressure acting on the genomes of BovHepV, and genetic recombination was not common among BovHepVs. These results expand the knowledge about the genetic diversity and evolution of BovHepV distributed globally, and also indicate genetically divergent BovHepV strains were co-circulating in cattle populations in China.     

Chemical sensing in mammalian host–bacterial commensal associations

The mammalian gastrointestinal (GI) tract is colonized by a complex consortium of bacterial species. Bacteria engage in chemical signaling to coordinate population-wide behavior. However, it is unclear if chemical sensing plays a role in establishing mammalian host–bacterial commensal relationships. Enterohemorrhagic Escherichia coli (EHEC) is a deadly human pathogen but is a member of the GI flora in cattle, its main reservoir. EHEC harbors SdiA, a regulator that senses acyl-homoserine lactones (AHLs) produced by other bacteria. Here, we show that SdiA is necessary for EHEC colonization of cattle and that AHLs are prominent within the bovine rumen but absent in other areas of the GI tract. We also assessed the rumen metagenome of heifers, and we show that it is dominated by Clostridia and/or Bacilli but also harbors Bacteroidetes. Of note, some members of the Bacteroidetes phyla have been previously reported to produce AHLs. SdiA-AHL chemical signaling aids EHEC in gauging these GI environments, and promotes adaptation to a commensal lifestyle. We show that chemical sensing in the mammalian GI tract determines the niche specificity for colonization by a commensal bacterium of its natural animal reservoir. Chemical sensing may be a general mechanism used by commensal bacteria to sense and adapt to their mammalian hosts. Additionally, because EHEC is largely prevalent in cattle herds, interference with SdiA-mediated cattle colonization is an exciting alternative to diminish contamination of meat products and cross-contamination of produce crops because of cattle shedding of this human pathogen.     

Virome Characterization in Commercial Bovine Serum Batches—A Potentially Needed Testing Strategy for Biological Products

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies’ established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols.     

Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis)

Isolation and characterization of circular replicase-encoding single-stranded (ss) DNA from animal, plant and environmental samples are rapidly evolving in virology. We detected 21 circular DNA elements, including one genomoviral sequence, in individual milk samples from domesticated Asian water buffaloes (Bubalus arnee f. bubalis). Most of the obtained genomes are related to Sphinx 1.76 and Sphinx 2.36 sequences and share a high degree of similarity to recently published circular DNAs—named BMMF (bovine meat and milk factors)—that have been isolated from commercial milk, as well as from bovine serum. Characteristic features such as rep genes, tandem repeats and inverted repeats were detected. These BMMF have recently been found to be present in taurine-type dairy cattle breeds descending from the aurochs (Bos primigenius). Importantly, the occurrence of BMMF has been linked to the higher incidence of colorectal and breast cancer in North America and Western Europe compared with Asia. This is the first report of circular ssDNA detected in milk from the domesticated form of the wild Asian water buffalo (B. arnee) belonging to the subfamily Bovinae. This novelty should be taken into account in view of the above-mentioned cancer hypothesis.     

Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host–pathogen interactions and malaria virulence.     

Melatonin improves the fertilization capacity and developmental ability of bovine oocytes by regulating cytoplasmic maturation events

Melatonin is a well‐characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10^–7, 10^–9, and 10^?11 mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CG s], and endoplasmic reticulum [ER ]), intracellular glutathione (GSH ) and ATP levels, expression of endogenous antioxidant genes (Cat , Sod1 , and GP x ), and fertilization‐related events (IP 3R1 distribution and expression of CD 9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization‐related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.     

Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB⁺). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I⁻ polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.     

Use of DNA restriction fragment typing in the differentiation of Mycobacterium tuberculosis complex isolates from animals and humans in Burundi

Setting: Bubanza Hospital and Veterinary Laboratory in Bujumbura, Burundi.Objective: To monitor the rate of Mycobacterium bovis infections among tuberculosis (TB) patients and among slaughtered cattle, and to analyse the polymorphism among deoxyribonucleic acid (DNA) fingerprints of the M. tuberculosis complex isolates.Design: 135 lymph node biopsies and 35 sputum specimens from human patients, together with specimens from 46 healthy animals and 36 animals suspected for bovine tuberculosis (BTB), were cultured. Isolates were identified phenotypically and DNA fingerprints were obtained by IS6110 based restriction fragment length polymorphism.Results: 119 M. tuberculosis complex isolates were obtained from 170 human specimens. M. bovis was not identified in any human sample. One out of 46 healthy animals and 14 out of 36 BTB suspected animals yielded M. bovis isolates. DNA fingerprinting revealed four to eight copies of IS6110 for all M. bovis isolates with some degree of polymorphism, and some clustering for human TB isolates. No relationship was observed between human and bovine isolates.Conclusion: At present M. bovis seems to play a minor role in human TB in Burundi, despite the high prevalence of both human immunodeficiency virus infection in humans and M. bovis in cattle. DNA fingerprinting is able to differentiate between bovine isolates.