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1.  BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm 
Retrovirology  2010;7:91.
Bovine leukemia virus (BLV) is closely related to human T-cell leukemia virus (HTLV) and is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly extended course that often involves persistent lymphocytosis and culminates in B-cell lymphomas. BLV provirus remains integrated in cellular genomes, even in the absence of detectable BLV antibodies. Therefore, to understand the mechanism of BLV-induced leukemogenesis and carry out the selection of BLV-infected animals, a detailed evaluation of changes in proviral load throughout the course of disease in BLV-infected cattle is required. The aim of this study was to develop a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in clinical animals.
Degenerate primers were designed from 52 individual BLV long terminal repeat (LTR) sequences identified from 356 BLV sequences in GenBank using the CoCoMo algorithm, which has been developed specifically for the detection of multiple virus species. Among 72 primer sets from 49 candidate primers, the most specific primer set was selected for detection of BLV LTR by melting curve analysis after real-time PCR amplification. An internal BLV TaqMan probe was used to enhance the specificity and sensitivity of the assay, and a parallel amplification of a single-copy host gene (the bovine leukocyte antigen DRA gene) was used to normalize genomic DNA. The assay is highly specific, sensitive, quantitative and reproducible, and was able to detect BLV in a number of samples that were negative using the previously developed nested PCR assay. The assay was also highly effective in detecting BLV in cattle from a range of international locations. Finally, this assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression.
Using our newly developed BLV-CoCoMo-qPCR assay, we were able to detect a wide range of mutated BLV viruses. CoCoMo algorithm may be a useful tool to design degenerate primers for quantification of proviral load for other retroviruses including HTLV and human immunodeficiency virus type 1.
PMCID: PMC2988707  PMID: 21044304
2.  Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV-CoCoMo-qPCR 
Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. BLV infection may remain clinically silent at the aleukemic (AL) stage, cause persistent lymphocytosis (PL), or, more rarely, B cell lymphoma. BLV has been identified in B cells, CD2+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, γ/δ T cells, monocytes, and granulocytes in infected cattle that do not have tumors, although the most consistently infected cell is the CD5+ B cell. The mechanism by which BLV causes uncontrolled CD5+ B cell proliferation is unknown. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method, BLV-CoCoMo-qPCR, which enabled us to demonstrate that the proviral load correlates not only with BLV infection, as assessed by syncytium formation, but also with BLV disease progression. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and BLV-CoCoMo-qPCR.
Phenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 × 105 cells identified a high percentage of CD5+ IgM+ cells (but not CD5- IgM+ B cells, CD4+ T cells, or CD8+T cells). These lymphocyte subpopulations were purified from three out of five cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5+ IgM+ B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5+ IgM+ B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells, and CD8+ T cells, even in BLV-infected cattle with a proviral load of <100 copies per 105 cells.
The results of the recent study showed that, although CD5+ IgM+ B cells were the main cell type targeted in BLV-infected but clinically normal cattle, CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells were infected to a greater extent than previously thought.
PMCID: PMC3648496  PMID: 23641811
Bovine leukemia virus (BLV); Proviral load; BLV-CoCoMo-qPCR; CD5+IgM+ B cell; Cell sorting; Flow cytometry
3.  Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction. 
Journal of Clinical Microbiology  1992;30(1):185-191.
Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine.
PMCID: PMC265018  PMID: 1370847
4.  Proviral detection and serology in bovine leukemia virus-exposed normal cattle and cattle with lymphoma. 
Twenty-seven cattle with lymphoma and 46 cows from a known bovine leukemia virus (BLV)-infected herd were tested for anti-BLV antibody by the agar gel immunodiffusion (AGID) test and an enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) and Southern hybridization were used to detect BLV provirus in the tumor DNA of the 27 cattle with lymphoma. The PCR was used to detect BLV provirus in the peripheral blood mononuclear cell DNA of the 46 normal known-exposed cattle. Two presumed false negative AGID test results compared to ELISA were found. Of ten cattle three years of age or less with "sporadic" forms of lymphoma, four had BLV provirus in tumor DNA, detectable by PCR. In two of these four, BLV provirus was clonally integrated based on digestion of tumor DNA with restriction enzymes followed by Southern hybridization. The BLV provirus was not detected by PCR in 5 of 17 cattle with "enzootic" lymphoma and two of these five were seronegative. Among normal BLV-exposed cows, 6.5% (3 of 46) were serologically positive and PCR negative; serologically negative and PCR positive cows occurred with the same frequency. Serological and PCR test results, when considered in all cattle (n = 73), had a concordance rate of 83.6%. Discordant test results occurred with approximately equal frequency between serologically positive and PCR negative (7 of 73, 9.6%) and serologically negative and PCR positive (5 of 73, 6.8%) groups. These data suggest that the role of BLV in some "sporadic" bovine lymphomas, previously unassociated with BLV, should be reexamined. The BLV provirus was not demonstrable in the tumor DNA from five adult cattle with lymphoma, suggesting that BLV may not be the etiological agent in all adult bovine lymphomas. The findings of persistently seronegative PCR positive and seropositive PCR negative cattle indicate that further work is needed to more fully understand the host-virus interaction. Present serological screening methods may not have sufficient sensitivity for determining BLV status in some circumstances.
PMCID: PMC1263566  PMID: 1335834
5.  In vivo leukocyte tropism of bovine leukemia virus in sheep and cattle. 
Journal of Virology  1994;68(7):4589-4596.
Bovine leukemia virus (BLV), an oncovirus related to human T-cell leukemia virus type I, causes a B-cell lymphoproliferative syndrome in cattle, leading to an inversion of the T-cell/B-cell ratio and, more rarely, to a B-cell lymphosarcoma. Sheep are highly sensitive to BLV experimental infection and develop B-cell pathologies similar to those in cattle in 90% of the cases. BLV tropism for B cells has been well documented, but the infection of other cell populations may also be involved in the BLV-induced lymphoproliferative syndrome. We thus looked for BLV provirus in other leukocyte populations in sheep and cattle by using PCR. We found that while B cells harbor the highest proviral load, CD8+ T cells, monocytes, and granulocytes, but not CD4+ T cells, also bear BLV provirus. As previously described, we found that persistent lymphocytosis in cows is characterized by an expansion of the CD5+ B-cell subpopulation but we did not confirm this observation in sheep in which the expanded B-cell population expressed the CD11b marker. Nevertheless, BLV could be detected both in bovine CD5+ and CD5- B cells and in sheep CD11b+ and CD11b- B cells, indicating that the restricted BLV tropism for a specific B-cell subpopulation cannot explain its expansion encountered in BLV infection. Altogether, this work shows that BLV tropism in leukocytes is wider than previously thought. These results lead the way to further studies of cellular interactions among B cells and other leukocytes that may intervene in the development of the lymphoproliferative syndrome induced by BLV infection.
PMCID: PMC236386  PMID: 8207833
6.  In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep. 
Journal of Virology  1991;65(5):2484-2490.
Expression of bovine leukemia virus (BLV) has been considered to be blocked at the transcriptional level in vivo, since viral RNA species are not readily detected in freshly isolated leukocytes from BLV-infected animals. However, the presence of a persistent antiviral antibody response in infected animals suggests that some degree of virus expression must occur in vivo. The purpose of this study was to determine whether BLV RNA species could be detected by using the polymerase chain reaction in normal or neoplastic lymphoid cells freshly isolated from naturally or experimentally BLV-infected cattle and sheep, respectively. Primers designed to detect a 2.1-kb doubly spliced BLV tax/rex-specific mRNA were used to amplify cDNA copies of RNA derived from infected animals. The amplified viral product was then detected with a radiolabeled BLV tax/rex-specific probe. BLV-specific RNA was detected readily in freshly isolated peripheral blood leukocytes derived from BLV-seropositive cattle or sheep with persistent lymphocytosis and less readily in peripheral blood leukocytes from BLV-seropositive but hematologically normal animals. BLV-specific RNA was also detected in fresh samples of BLV-induced lymphosarcomas. Normal and neoplastic lymphoid cells from BLV-seronegative animals were uniformly negative under similar conditions. These primers also amplified the same viral product from genomic DNA derived from BLV-seropositive animals, providing further evidence for in vivo transcription and suggesting that BLV RNA-dependent DNA polymerase is capable of reverse transcribing the 2.1-kb mRNA in vivo. The demonstration of transcriptional products of BLV in vivo proves that viral latency in BLV infection is incomplete.
PMCID: PMC240603  PMID: 1850025
7.  Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30T-) 
Journal of Veterinary Science  2009;10(4):331-336.
Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.
PMCID: PMC2807270  PMID: 19934599
AGID; baculovirus expression; bovine leukemia virus; glycoproteins
8.  Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model 
Journal of Virology  1999;73(10):8160-8166.
Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV.
PMCID: PMC112833  PMID: 10482566
9.  In vivo study of genetically simplified bovine leukemia virus derivatives that lack tax and rex. 
Journal of Virology  1997;71(2):1514-1520.
Genetically simplified derivatives of complex retroviruses that replicate in animal models are useful tools to study the role of the complex regulatory genes in virus infection and pathogenesis and were proposed as a novel approach toward the development of vaccines against complex retroviruses. Previously we developed genetically simple derivatives of bovine leukemia virus (BLV) that can replicate in tissue culture independently of the BLV regulatory proteins, Tax and Rex, and the RIII and GIV open reading frames (K. Boris-Lawrie and H. M. Temin, J. Virol. 69:1920-1924, 1995). These derivatives are encoded on novel, hybrid retrovirus genomes that contain transcriptional control sequences of a simple retrovirus and gag-pol or env genes of the complex BLV. The first-generation simple BLV derivatives replicate as complementary viruses (coviruses) by using separate gag-pol or env genomes, and therefore virus spread is limited to cells that are infected with both covirus genomes. Here we describe a second-generation simple BLV derivative that is encoded on a single hybrid genome. We show the virus to be replication competent by successive passage on D17 target cells and by analysis of viral RNA and proteins in the infected cells. Furthermore, we evaluate the immunogenicity and infectivity of the simple BLV derivatives in a BLV animal model. Small groups of rats were injected either with virus-producing cells or with proviral DNA. Western immunoblot analysis revealed that antibodies against the major viral antigenic determinants are induced in response to either method of introduction and that seroconversion is sustained in most of the rats for at least 6 months (the duration of the study). The magnitudes of the antiviral responses were similar in rats infected with the first-generation simple BLV coviruses, the second-generation replication-competent derivative, or wild-type BLV. Wild-type BLV typically infects peripheral blood mononuclear cells (PBMC), and the simple BLV derivatives were also found to infect PBMC as demonstrated by PCR amplification of proviral sequences and reverse transcriptase PCR amplification of viral RNA in treated rats. These results establish that simple BLV derivatives lacking tax and rex are infectious and immunogenic in rats. These viruses will be useful tools in comparative studies with BLV to evaluate the role of tax and rex in maintenance of virus load and in disease outcome.
PMCID: PMC191208  PMID: 8995677
10.  Unintegrated bovine leukemia virus DNA: association with viral expression and disease. 
Journal of Virology  1996;70(8):4961-4965.
The correlation between bovine leukemia virus (BLV) unintegrated DNA, viral expression, and stage of disease was determined in cattle naturally infected with BLV. The concomitant presence of unintegrated BLV DNA with viral transcriptional activity was observed in 53% (18 of 34) of hematologically normal, BLV-seropositive cattle and in 100% (10 of 10) of BLV-seropositive cattle with the preneoplastic syndrome persistent lymphocytosis. In vitro studies suggested that accumulation of unintegrated BLV DNA resulted from a process of reinfection rather than intracellular reverse transcription of newly synthesized BLV RNA. Interestingly, unintegrated BLV DNA was not detected in tumor cells from cattle with BLV-associated lymphocytic leukemia/malignant lymphoma despite viral transcriptional activity in 100% (eight of eight) of these cattle. Thus, the presence of unintegrated BLV DNA differentiated nonneoplastic from neoplastic conditions in BLV-infected cattle. These results demonstrate that unintegrated viral DNA serves as a marker of disease progression in BLV-infected cattle but is not necessarily associated with induction or maintenance of the neoplastic state.
PMCID: PMC190448  PMID: 8764001
11.  Bovine leukemia virus p24 antibodies reflect blood proviral load 
Bovine leukemia virus (BLV) is worldwide distributed and highly endemic in Argentina. Among the strategies to prevent BLV dissemination, a control plan based on the selective segregation of animals according to their proviral load (PVL) is promising for our dairy productive system. The objective of this work was to study the relationship between the blood PVL and the antibody level, in order to identify whether the individual humoral response, i.e. the anti-p24 or anti-whole-BLV particle, could be used as a marker of the blood level of infection and thus help to recruit animals that may pose a lower risk of dissemination under natural conditions.
The prevalence of p24 antibodies on the 15 farms studied was over 66%. The prevalence of p24 and whole-BLV antibodies and PVL quantification were analyzed in all the samples (n = 196) taken from herds T1 and 51. ROC analysis showed a higher AUC for p24 antibodies than whole-BLV antibodies (Zreactivity: 3.55, P < 0.001; Ztiter: 2.88, P < 0.01), and as consequence a better performance to predict the proviral load status in herd 51. No significant differences were found between the performance of p24 and whole-BLV antibodies in herd T1. A significant positive correlation was observed between PVL values and p24 antibody reactivity in both farms (r T1 = 0.7, P < 0.001, r 51 = 0.71, P < 0.0001). The analysis was extended to the whole number of weak p24 antibody reactors (n = 311) of the other 13 farms. The mean of high PVL reactors within weak p24 reactors was 17.38% (SD = 8.92). In 5/15 farms, the number of weak p24 reactors with high PVL was lower than 10%.
We found that the humoral response reflected the level of in vivo infection, and may therefore have useful epidemiological applications. Whereas the quantitative evaluation of blood proviral load using real-time PCR is expensive and technically demanding, the measurement of antibodies in blood by ELISA is relatively straightforward and could therefore constitute a cost-effective tool in a BLV control intervention strategy, especially in highly infected herds such as Argentinean dairy ones.
PMCID: PMC3526540  PMID: 23047073
BLV; Proviral load; Control measures; Serological marker; ROC curve; p24 antibodies
12.  Role of the Proline-Rich Motif of Bovine Leukemia Virus Transmembrane Protein gp30 in Viral Load and Pathogenicity in Sheep 
Journal of Virology  2001;75(17):8082-8089.
The cytoplasmic tail of bovine leukemia virus (BLV) transmembrane protein gp30 has multiple amino acid motifs that mimic those present in signaling proteins associated with B-cell and T-cell receptors. The proline-rich motif of gp30, PX2PX4–5P, is analogous to the recognition site of Src homology 3 (SH3) domains of signaling molecules. Using site-directed mutagenesis of an infectious molecular clone of BLV, point mutations were introduced which changed three of the prolines of the motif to alanines. The influence of these mutations on the pathogenicity of BLV was studied in sheep which received either (i) plasmid DNA with provirus containing proline-to-alanine mutations (pppBLV), (ii) plasmid DNA with wild-type provirus (wtBLV), or (iii) transfection reagent alone. Although all of the BLV-injected animals seroconverted at approximately the same time, viral loads at later time points were high in five of five of the wtBLV group and two of five of the pppBLV group but low in three of five of the pppBLV group, as determined by semiquantitative PCR. Viral expression was lower in the pppBLV-transfected sheep, as measured by p24 antigen enzyme-linked immunosorbent assay in cultured cells, and serologic titers were lower. Thirty-one months after transfection, four of four wtBLV-transfected sheep had died of leukemia and lymphoma, and all five of the pppBLV-transfected sheep were clinically healthy and had normal peripheral blood lymphocyte counts. These data indicate that the proline-rich motif of gp30 is not required for viral infectivity but is important for high viral load in vivo, suggesting that SH3-mediated gp30 interactions are critical for viral pathogenesis following infection. Absence of interactions with the proline-rich motif may prevent or delay tumorigenesis in sheep.
PMCID: PMC115052  PMID: 11483753
13.  Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10 
Journal of Virology  1999;73(10):8427-8434.
Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.
PMCID: PMC112861  PMID: 10482594
14.  Complete Bovine Leukemia Virus (BLV) Provirus Is Conserved in BLV-Infected Cattle throughout the Course of B-Cell Lymphosarcoma Development 
Journal of Virology  1998;72(9):7569-7576.
Bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) belong to the same subfamily of oncoviruses. Defective HTLV-1 proviral genomes have been found in more than half of all patients with adult T-cell leukemia examined. We have characterized the genomic structure of integrated BLV proviruses in peripheral blood lymphocytes and tumor tissue taken from animals with lymphomas at various stages. Genomic Southern hybridization with SacI, which generates two major fragments of BLV proviral DNA, yielded only bands that corresponded to a full-size provirus in all of 23 cattle at the lymphoma stage and in 7 BLV-infected but healthy cattle. Long PCR with primers located in long terminal repeats clearly demonstrated that almost the complete provirus was retained in all of 27 cattle with lymphomas and in 19 infected but healthy cattle. However, in addition to a PCR product that corresponded to a full-size provirus, a fragment shorter than that of the complete virus was produced in only one of the 27 animals with lymphomas. Moreover, when we performed conventional PCR with a variety of primers that spanned the entire BLV genome to detect even small defects, PCR products were produced that specifically covered the entire BLV genome in all of the 40 BLV-infected cattle tested. Therefore, it appears that at least one copy of the full-length BLV proviral genome was maintained in each animal throughout the course of the disease and, in addition, that either large or small deletions of proviral genomes may be very rare events in BLV-infected cattle.
PMCID: PMC110004  PMID: 9696855
15.  The Region between Amino Acids 245 and 265 of the Bovine Leukemia Virus (BLV) Tax Protein Restricts Transactivation Not Only via the BLV Enhancer but Also via Other Retrovirus Enhancers 
Journal of Virology  2000;74(23):10939-10949.
Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type 1 (HTLV-1). The Tax protein of BLV acts through the 5′ long terminal repeat (LTR) of BLV and activates the transcription of BLV. In this study, we amplified tax genes from BLV-infected cattle using PCR. We cloned the genes and monitored the transcriptional activities of the products. Seven independent mutant Tax proteins, with at least one amino acid substitution between residues 240 and 265, exhibited a markedly stronger ability to stimulate the viral LTR-directed transcription than the wild-type Tax protein. Analysis of chimeric Tax proteins derived from wild-type and mutant Tax proteins clearly demonstrated that a single substitution between residue 240 and 265 might be critical for the higher activities of the Tax mutant proteins. Furthermore, it appeared that transient expression of a Tax mutant protein was better able to increase the production of viral proteins and particles from a defective recombinant proviral clone of BLV than was wild-type Tax. Analysis of mutations within the U3 region of the LTR revealed that a cyclic AMP-responsive element in Tax-responsive element 2 might be sufficient for the enhanced activation mediated by the mutant proteins. In addition to the LTR of BLV, other viral enhancers, such as the enhancers of HTLV-1 and of mouse mammary tumor virus, which cannot be activated by wild-type BLV Tax protein, were activated by a Tax mutant protein. Our observations suggest that the transactivation activity and target sequence specificity of BLV Tax might be limited or negatively regulated by the region of the protein between amino acids 240 and 265.
PMCID: PMC113173  PMID: 11069988
16.  Cell Infectivity in Relation to Bovine Leukemia Virus gp51 and p24 in Bovine Milk Exosomes 
PLoS ONE  2013;8(10):e77359.
Exosomes are small membranous microvesicles (40–100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env) and p24 (Gag), were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells.
PMCID: PMC3798320  PMID: 24146982
17.  The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions 
Journal of Virology  1999;73(2):1293-1301.
The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.
PMCID: PMC103953  PMID: 9882334
18.  Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70. 
Journal of Virology  1993;67(4):1803-1810.
We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.
PMCID: PMC240230  PMID: 7680387
19.  Massive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites during Primary Infection 
PLoS Pathogens  2013;9(10):e1003687.
Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.
Author Summary
Human T-lymphotropic Virus 1 (HTLV-1) induces a persistent infection that remains generally asymptomatic. Nevertheless, in a small proportion of individuals and after a long latency, HTLV-1 infection leads to leukemia or lymphoma. Onset of clinical manifestations correlates with a persistently elevated number of infected cells. Because the vast majority of cells are infected at early stages, primary infection is a crucial period for HTLV-1 persistence and pathogenesis. Since HTLV-1 is transmitted through breast feeding and because systematic population screenings are rare, there is a lack of available samples at early infection. Therefore, we addressed this question in a closely related animal model by inoculating cows with Bovine Leukemia Virus (BLV). We show that the vast majority of cells becoming infected during the first weeks of infection and do not survive later on. We also demonstrate that the initial host selection occurring during primary infection will specifically target cells that carry a provirus inserted in genomic transcribed regions. This conclusion thus highlights a key role exerted by the host immune system during primary infection and indicates that antiviral treatments would be optimal when introduced straight after infection.
PMCID: PMC3789779  PMID: 24098130
20.  Pathogenicity of molecularly cloned bovine leukemia virus. 
Journal of Virology  1993;67(12):7096-7105.
To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.
PMCID: PMC238171  PMID: 8230433
21.  Inhibition of protein kinase C results in decreased expression of bovine leukemia virus. 
Journal of Virology  1992;66(7):4427-4433.
The in vitro expression of bovine leukemia virus (BLV) in short-term cultured bovine peripheral blood mononuclear cells (PBMC) is associated with increased spontaneous lymphocyte blastogenesis. The purpose of this study was to determine whether intracellular pathways responsible for antigen- or mitogen-induced lymphocyte blastogenesis were also responsible for induction of BLV expression. The protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride (3-methyl H7) decreased blastogenesis in a dose-dependent manner, as measured by [3H]thymidine incorporation, in unstimulated, lipopolysaccharide-stimulated and phorbol ester (PMA)-stimulated BLV-infected PBMC. Similarly, 3-methyl H7 decreased BLV expression, as measured by production of gp51 envelope antigen or p24gag antigen, in BLV-infected PBMC under the same conditions. Using an RNase protection assay, the inhibition of BLV expression by 3-methyl H7 was shown to be due to decreased transcriptional activity. The cyclic GMP-dependent protein kinase and cyclic AMP-dependent protein kinase inhibitor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) did not inhibit either BLV expression or blastogenesis of BLV-infected bovine PBMC. Additional evidence for the PKC-dependent expression of BLV was obtained by using a persistently BLV-infected B-lymphocyte cell line, NBC-13. Activation of PKC by PMA in NBC-13 cells increased BLV expression. 3-methyl H7 decreased the PMA-induced expression of BLV in NBC-13 cells in a dose-dependent manner, whereas HA1004 did not inhibit this expression. These results identify a mechanism for the induction of BLV expression through PKC activation and therefore indicate that latency and replication of BLV is controlled by normal B-lymphocyte intracellular signaling pathways.
PMCID: PMC241250  PMID: 1318412
22.  A Mutant Form of the Tax Protein of Bovine Leukemia Virus (BLV), with Enhanced Transactivation Activity, Increases Expression and Propagation of BLV In Vitro but Not In Vivo 
Journal of Virology  2003;77(3):1894-1903.
In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.
PMCID: PMC140974  PMID: 12525624
23.  Investigation of the Bovine Leukemia Virus Proviral DNA in Human Leukemias and Lung cancers in Korea 
Journal of Korean Medical Science  2005;20(4):603-606.
The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans.
PMCID: PMC2782155  PMID: 16100451
Leukemia; Lung Neoplasms; Leukemia Virus, Bovine; Human Infection
24.  Dynamics of perinatal bovine leukemia virus infection 
Bovine leukemia virus (BLV) is highly endemic in many countries, including Argentina. As prevention of the spread from infected animals is of primary importance in breaking the cycle of BLV transmission, it is important to know the pathophysiology of BLV infection in young animals, as they are the main source of animal movement. In this work, we determined the proviral load and antibody titers of infected newborn calves from birth to first parturition (36 months).
All calves under study were born to infected dams with high proviral load (PVL) in blood and high antibody titers and detectable provirus in the colostrum. The PVL for five out of seven calves was low at birth. All animals reached PVLs of more than 1% infected peripheral blood mononuclear cells (PBMCs), three at 3 months, one at 6 months, and one at 12 months. High PVLs persisted until the end of the study, and, in two animals, exceeded one BLV copy per cell. Two other calves maintained a high PVL from birth until the end of the study. Antibody titers were 32 or higher in the first sample from six out of seven calves. These decayed at 3–6 months to 16 or lower, and then increased again after this point.
Calves infected during the first week of life could play an active role in early propagation of BLV to susceptible animals, since their PVL raised up during the first 12 months and persist as high for years. Early elimination could help to prevent transmission to young susceptible animals and to their own offspring. To our knowledge, this is the first study of the kinetics of BLV proviral load and antibody titers in newborn infected calves.
PMCID: PMC3986441  PMID: 24708791
BLV; Perinatal infection; Proviral load; Natural infection
25.  Agar gel immunodiffusion test for the detection of bovine leukemia virus antibodies: lack of trans-Atlantic standardization. 
Two agar gel immunodiffusion (AGID) kits for the serodiagnosis of bovine leukemia virus (BLV) were imported from Europe and were compared with North American kits. The BLV AGID kits from North America and from Europe differed significantly. The punches were different, as were the pattern distribution in the agar of the reference and the test sera, resulting in differences in the reading of the immunoprecipitation lines. Based on the testing of 1200 serum samples from cattle, the European kits gave a good correlation with the American kits, as indicated by their respective kappa values. However, the European kits were found to be less sensitive when evaluated against weakly positive samples from field specimens or following a dilution trial. Only 65% and 50% of the weakly positive samples detected by the American kit #1 were detected by the European kits #2 and #3, respectively. The American kit was also capable of detecting BLV antibodies in 45% of strongly positive samples diluted 1/50 in negative sera, while antibodies were detected in only 15% of the samples with the European kit #2 and in none of the samples with the European kit #3. False negatives were also detected with the European kits. Among the false negatives, the degree of expected reactions was weak (European kit #2) or of varying degrees of positivity (European kit #3). Besides the differences in format and performance, the BLV-AGID kits in Europe are evaluated with the National Standard Serum E4 while a proficiency panel composed of a quadruplicate set of 10 reference sera is used in Canada to monitor the kits. Based on the overall observations, we noted a lack of standardization between the BLV-AGID kits used in North America and in Europe.
PMCID: PMC1189591  PMID: 10805247

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