BLV-infected Cattle Research Articles

Bovine MHC class II DR molecule plays a key role in bovine leukemia virus (BLV)-induced lymphoma

BLV is the etiological agent of enzootic bovine leukosis (EBL), which emerges as B-cell lymphomas. Previously, we found one of tumor-associated antigens (TAAs), which is a useful tool not only for diagnosing EBL but also for screening of BLV-infected cattle with the potential to develop tumor in the future, by using the monoclonal antibody (MAb) c143. We here performed biochemical characterization of c143 TAA such as two-dimensional electrophoresis, sequential immunoprecipitation and tryptic peptide mapping with c143 MAb as well as MAbs against bovine major histocompatibility complex (BoLA) class II antigens, and flow cytometry and immunofluorescence staining of cells transiently transfection with a combination of BoLA-DRA, -DRB3, -DQA and -DQB cDNAs. First, it was found that the c143 TAA is BoLA-DR consisting of alpha and beta chains. Furthermore, the altered expressions of c143 TAA were observed in infected animal with lymphoma at various stages as follows: 1) The number of cells positive for the c143 TAA and 2) glycosylation of the beta chain increased with the progression of EBL. 3) The beta chain was specifically phosphorylated at serine residue(s) during the lymphoma stage. Finally, we tested an association of microsatellite markers using 86 cattle with lymphoma and 320 asymptomatic cattle. We identified that seven genomic markers which located in chromosomes 1, 3, 9, 15 , 21, 23 and 24, and, in addition, BoLA-DRB3 gene in chromosomes 23, showed suggestive linkage to development of the BLV-induced EBL. Collectively, our results strongly indicate that BoLA-DR plays a key role in BLV-induced diseases progression.

BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

BackgroundBovine 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.ResultsDegenerate 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.ConclusionsUsing 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.

Association of BoLA-DRB3.2 Alleles with Resistance and Susceptibility to Persistent Lymphocytosis in BLV Infected Cattle in Argentina

Fil: Panei, Carlos Javier. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiologia. Catedra de Virologia; Argentina. Provincia de Buenos Aires. Gobernacion. Comision de Investigaciones Cientificas; Argentina

Host soluble factors that regulate the synthesis of the major core protein of the bovine leukemia virus (BLV) in a naturally infected neoplastic B-cell line

Bovine leukemia virus (BLV) is a B-cell tropic Deltaretrovirus that induces a lifelong infection and causes a fatal lymphosarcoma in less than 10% of the infected cattle. BLV is usually present in its host in a transcriptional repressed state but becomes de-repressed a few hours after the infected lymphocytes are cultured in vitro. In the present study we have examined the effect of soluble host factors and various substances on the synthesis of the major BLV protein (p24) in a permanent culture (cell line NBC-10) of neoplastic B-lymphocytes derived from BLV-infected cattle. Certain batches of fetal calf serum (FCS) and bovine platelet lysates (PLy) induced a rapid and drastic increase of the synthesis of BLVp24 in the NBC-10 cells. Neutralization experiments with specific antibodies demonstrated that the transforming growth factor-β (TGF-β) was responsible for the stimulatory activity of FCS and PLy on the synthesis of BLVp24 in the NBC-10 cells. Recombinant TGF-β also stimulated the synthesis of BLVp24 in cultures of peripheral blood mononuclear cells (PBMCs) obtained from BLV-infected cattle. Mitogens, phorbol-myristate-acetate and prostaglandin E 2, previously shown to stimulate the expression of BLV in cultures of PBMC, did not induce the synthesis of BLVp24 in cultures of NBC-10 cells. Plasma, serum and milk from BLV-negative cattle inhibited the synthesis of BLVp24 induced by FCS, PLy or TGF-β in the NBC-10 cells. The blocking activity was found in the whey and the β-casein fractions of bovine milk. The relevance of these findings with regard to the previously reported plasma factor (PBB) with blocking activity on the expression of BLV in short-term PBMC cultures is discussed. Based on the information obtained in the present study we have standardized a reproducible and rapid assay system for the identification of factors that regulate the synthesis of BLVp24 in naturally infected neoplastic B cells.

Association of BLV infection profiles with alleles of the BoLA‐DRB3.2 gene

Bovine leukaemia virus (BLV) causes lymphosarcoma and persistent lymphocytosis (PL). Some MHC class II gene polymorphisms have been associated with resistance and susceptibility to the development of lymphosarcoma and PL, as well as with a reduced number of circulating BLV-infected lymphocytes. Previously, 230 BLV-infected Holstein cattle were classified into two infection profiles characterized by low and high proviral loads (LPL and HPL respectively). Here, the influence of the polymorphism at the BoLA-DRB3.2* gene of these animals was examined. After genotyping, the association between the BoLA-DRB3.2* alleles and the BLV infection profile was determined as the odds ratio (OR). Two subtypes of allele *11 were identified (ISAG*0901 and *0902). Allele ISAG*0902 showed a stronger association with the LPL profile (OR = 8.24; P < 0.0001) than allele *11 itself (OR = 5.82; P < 0.0001). Allele ISAG*1701 (*12) also showed significant association with the LPL profile (OR = 3.46; P < 0.0055). Only one allele, ISAG*1501 or 03 (*16), showed significant association with HPL (OR = 0.36; P < 0.0005). The DRB3.2* alleles were assigned to three categories: resistant (R), susceptible (S) and neutral (N). Based on their DRB3 genotypes, cattle were classified as homozygous or heterozygous. The RR and RN genotypes were associated with the LPL profile, while the SS and NS genotypes were associated with the HPL profile. The RS genotype could not be associated with any particular profile. Our results show that allele ISAG*0902 appears to be the best BLV resistance marker in Holstein cattle.

Amperometric immunosensor for diagnosis of BLV infection

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody–antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N′,N′-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.

Determination of proviral load in bovine leukemia virus–infected cattle with and without lymphocytosis

To determine proviral load in bovine leukemia virus (BLV)-infected cattle with and without persistent lymphocytosis to assess the potential of transmitting the virus. Cattle in 6 dairy herds. Blood samples from infected cows were evaluated 3 times at 6-month intervals for determination of proviral load via PCR assay, serologic results via ELISA, and hematologic status via differential cell counts. Infected cattle were classified into lymphocytotic and nonlymphocytotic groups. Lymphocytotic cattle consistently had > 100,000 copies of integrated provirus/mug of DNA (ie, high proviral load) in peripheral blood leukocytes. Titers of antibodies against BLVgp51 and BLVp24 indicated a strong immune response. Nonlymphocytotic cattle comprised 2 subgroups: a group with high proviral load and strong immune response, and a group with a weaker immune response, mostly against BLVp24, and a proviral load of < 100 copies/microg of DNA (ie, low proviral load). Results emphasized the importance of characterizing nonlymphocytotic BLV-infected cattle during eradication programs. The risk of transmitting BLV infection from nonlymphocytotic cattle may differ depending on the proviral load. Nonlymphocytotic cattle with high proviral load could be efficient transmitters (as efficient as lymphocytotic cattle), whereas nonlymphocytotic cattle with low proviral load could be inefficient transmitters under standard husbandry conditions. Because most cattle with low proviral load do not develop anti-BLVp24 antibodies, it appears that lack of an anti-BLVp24 antibody response may be a good marker of this condition.

In Vitro and In Vivo Effects of Recombinant Bovine Interferon-.TAU. on Bovine Leukemia Virus

The antiviral effects of recombinant bovine interferon-tau (rboIFN-tau) on bovine leukemia virus (BLV) were examined in vitro and in vivo. In the in vitro experiments, BLV titers decreased in FLK-BLV cells and in peripheral blood mononuclear cells of BLV-infected cattle treated with rboIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the in vivo effects of rboIFN-tau, 10 BLV-infected cattle were subcutaneously injected with rboIFN-tau. In the first experiment, 6 cows were administrated with 10(5) U/kg body weight of rboIFN-tau 3 times per week for 4 weeks, while in the second experiment 4 cows were administrated with 10(6) U/kg body weight of rboIFN-tau 3 times per week for 3 weeks. No adverse effects were observed after the administration of rboIFN-tau. In experiment No. 1, the mean BLV titers in cattle decreased in the post-rboIFN-tau administration period compared to the pre-rboIFN-tau administration period. In experiment No. 2, the mean BLV titers in cattle decreased in the rboIFN-tau administration period. These results suggest that rboIFN-tau decreases BLV titers in vitro and in vivo and that rboIFN-tau possibly reduces the degree of BLV titer in cattle without severe side effects.

Tumor necrosis factor-alpha up-regulation in spontaneously proliferating cells derived from bovine leukemia virus-infected cattle

We previously reported that tumor necrosis factor alpha (TNF-alpha) was one of the cytokines that contributed to the leukemogenesis caused by bovine leukemia virus (BLV). To determine if the spontaneous cell proliferation observed in the late disease stages, such as persistent lymphocytosis and lymphosarcoma, correlated with the expression level of TNF-alpha, we analyzed the mRNA expression levels for TNF-alpha in spontaneously proliferating PBMCs derived from BLV-infected cattle. The mean mRNA expression level for TNF-alpha was higher in the spontaneously proliferating PBMCs derived from BLV-infected cattle than in non-spontaneously proliferating PBMCs from normal cattle. The TNF-alpha protein level in the PBMCs was determined by flow cytometric analysis, and it was noted that most of the cells expressing membrane-bound TNF-alpha in the spontaneously proliferating cells were CD5+ or sIgM+-cells. Additionally, in order to determine if this spontaneous proliferation can be blocked by anti-bovine TNF-alpha MAb, the spontaneously proliferating PBMCs from a BLV-infected cattle were cultured in the presence of the MAb. The addition of this MAb at the beginning of the 72 h-cultivation clearly inhibited spontaneous proliferation of cells in a dose-dependent manner, indicating the direct involvement of TNF-alpha in the spontaneous proliferation of PBMCs during the late disease stage. These data suggest that an aberrant expression of TNF-alpha might contribute to the progression of bovine leukosis in animals which develop persistent lymphocytosis of B-cells or B-cell lymphosarcoma.

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-α and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-α-induced responses, in this study we examined the TNF-α-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-α (rTNF-α) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5 + or sIgM + cells and these cells showed resistance to TNF-α-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-α-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.

Isolation of a bovine plasma fibronectin-containing complex which inhibits the expression of bovine leukemia virus p24.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the "plasma blocking factor" (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65 degrees C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.

Induction of expression of bovine leukemia virus (BLV) in blood taken from BLV-infected cows without removal of plasma

The expression of bovine leukemia virus (BLV) is blocked at the transcriptional level during the so-called latency period. However, when peripheral blood mononuclear cells and B lymphocytes are isolated from BLV-infected animals and incubated in the presence of activating reagents, such as phorbol ester, the expression of BLV is markedly enhanced. Such “reactivation” is thought to play a crucial role in the spread of BLV from infected to uninfected cattle. In the present study, we found that the expression of BLV in samples of whole blood from BLV-infected cattle was activated immediately upon incubation at 37 °C and that such activation did not require the addition of any exogenous factors except for anticoagulants or the removal of blood cells from plasma. The expression of BLV was repressed by an inhibitor of protein kinase C (PKC), namely, H-7, and by a membrane-permeable chelator of Ca 2+ ions, BAPTA/AM. We also found that several isotypes of PKC were translocated immediately from the cytoplasm to the membrane fraction upon incubation of whole blood at 37 °C. Our data suggest that the actual collection of blood and pathways that involve PKC and Ca 2+ might play important roles in the reactivation of expression of BLV in blood from infected cattle.

VALIDAÇÃO DE NESTED REAÇÃO EM CADEIA DA POLIMERASE PARA DETECÇÃO DO VÍRUS DA LEUCOSE BOVINA EM AMOSTRAS DE SANGUE BOVINO DE REBANHO LEITEIRO: COMPARAÇÃO COM ENSAIO IMUNOENZIMÁTICO E IMUNODIFUSÃO EM GEL DE ÁGAR

ABSTRACT A sensitive and specific nested polymerase chain reaction (nested PCR) for detection of bovine leukemia virus (BLV) in blood samples was evaluated as a reliable assay for accurate diagnosis of naturally infected animals under field conditions. The standardized nested PCR relies on the identification of BLV DNA fragments of 340 or 444 bp produced by applying 2 different pairs of primers sets to the proviral env gene. This protocol was validated by testing 40 bovine blood samples from a BLV-positive dairy herd, and by comparing results with serological diagnosis provided by agar-gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). The nested PCR protocol was able to discriminate infected animals from that ones serodiagnosed as negative. This method also showed applicability for other body fluids or tissue samples. Investigation of 40 blood samples from a dairy herd showed that 37 animals were positive in ELISA, 36 in nested PCR, and only 25 in AGID. The nested PCR has demonstrated a good agreement in the results with ELISA up to 95%. These results demonstrated that nested PCR could be indicated for an early, sensitive and direct detection of BLV-infection in naturally infected cattle. Compared to the serological tests, this method proved to be the most suitable for identification of BLV-infected cattle with low, transient or without BLV-antibody titers. Moreover, this assay can be used to confirm or exclude BLV-infection in asymptomatic cattle or with doubtful serological results. This nested PCR is a valuable assay for prompt epidemiological investigations of BLV as well as for surveillance of animal health for bovine leukosis.

Increase in gammadelta T cells in the blood of cattle persistently infected with bovine leukemia virus following administration of recombinant bovine IFN-gamma.

To examine the effect of recombinant bovine interferon-gamma (rbIFN-gamma) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-gamma. All cattle were febrile after inoculation with IFN-gamma and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4+ and CD8+ T cells were decreased for 2-3 days and then their numbers were recovered. The number of gammadelta T cells increased after the fever. In contrast, the number of IgM+ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-gamma administration. These results suggest that IFN-gamma induces the up-regulation of gammadelta T cells, decreases the number of IgM+ lymphocytes and suppresses the growth of BLV in BLV-infected cattle in vivo.

Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle

Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5 +, IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5 + B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis.

Latency of viral expression in vivo is not related to CpG methylation in the U3 region and part of the R region of the long terminal repeat of bovine leukemia virus.

Bovine leukemia virus (BLV) is silent in most cells detectable in vivo, and the repression of its expression allows BLV to evade the host's immune response. In this study, we examined whether CpG methylation of DNA might be involved in the regulation of the expression of BLV in vivo. To investigate the effects of CpG methylation on the activity of the long terminal repeat (LTR) of BLV, we measured the transactivation activity of this region after treatment with the CpG methyltransferase SssI by using a luciferase reporter system. The activity of methylated LTR was significantly lower than that of nonmethylated LTR. Therefore, we examined the extent of CpG methylation of the U3 region and part of the R region of the LTR in BLV-infected cattle and in experimentally BLV-infected sheep at various clinical stages by the bisulfite genomic sequencing method. We detected no or minimal CpG methylation at all stages examined in cattle and sheep, and our results indicate that CpG methylation probably does not participate in the silencing of BLV in vivo.

Reduced IL-2 and IL-4 mRNA Expression in CD4 + T Cells from Bovine Leukemia Virus-Infected Cows with Persistent Lymphocytosis

The role of T-helper (Th) responses in the subclinical progression of bovine leukemia virus (BLV) infection was explored by determining the contribution of CD4 + T cells to the expression of mRNAs encoding interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) in BLV-infected cattle. Relative levels of mRNA encoding IFN-γ, IL-2, IL-4, and IL-10 were measured in fresh and concanavalin A (Con A) activated peripheral blood mononuclear cells (PBMCs) and purified CD4 + T cells from cows seronegative to BLV (BLV −), seropositive without persistent lymphocytosis (BLV +PL −), and seropositive with PL (BLV +PL +) using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The expressions of IFN-γ, IL-2, and IL-4 mRNAs were significantly reduced in the PBMCs from BLV +PL + cows as compared to BLV − cows. Reduced levels of IL-2 and IL-4 mRNAs were detected in fresh CD4 + T cells from BLV +PL + cows. In contrast, Con A stimulated PBMCs and CD4 + T cells did not differ significantly in expression of IFN-γ, IL-2, IL-10, or IL-4 mRNAs among the BLV infection groups. Using flow-sorted CD4 + T cells and semiquantitative RT-PCR the frequencies of CD4 + T cells transcribing IFN-γ, IL-2, IL-4, and IL-10 mRNAs in the peripheral blood of BLV −, BLV +PL −, and BLV +PL + cows were determined. There were no significant differences in the frequencies of CD4 + T cells expressing these cytokine mRNAs among animals in the different BLV infection categories. Thus, the observed differences in IL-2 and IL-4 mRNAs in CD4 + T cells were due to changes in steady-state mRNA levels expressed by individual cells and not to changes in the frequency of cells transcribing IL-2 and IL-4 mRNAs. These results demonstrate that the progression of BLV infection to PL is associated with reduced expression of classical Th1 and Th2 cytokines by CD4 + T cells, thus suggesting aberrant Th regulation in subclinically infected animals.

CD5 is dissociated from the B-cell receptor in B cells from bovine leukemia virus-infected, persistently lymphocytotic cattle: consequences to B-cell receptor-mediated apoptosis.

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5(+) B-cell population, termed persistent lymphocytosis (PL). As in human CD5(+) B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5(+) B cells. In contrast, in CD5(+) B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5(+) B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5(+) B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.

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.

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.

CD4 T lymphocyte activation in BLV-induced persistent B lymphocytosis in cattle.

Bovine leukemia virus (BLV) is an oncogenic retrovirus in the human T cell leukemia virus family. BLV infects B lymphocytes and induces a nonmalignant persistent lymphocytosis (PL) and leukemia/lymphoma in cattle. There is evidence that CD4 T lymphocytes are activated during BLV infection and promote the development of PL. How CD4 T lymphocytes are activated by BLV infection is not known. We observed that CD4 T lymphocytes from PL cattle proliferated in the presence of autologous, irradiated peripheral blood mononuclear cells (PBMC), whereas no proliferation occurred in cell cultures from BLV-infected non-PL cattle. Proliferation required direct contact with metabolically active irradiated PBMC but was not associated with viral protein expression or inhibited by antibodies to BLV. Unexpectedly, B lymphocytes alone failed to account for the irradiated PBMC stimulation of CD4 T lymphocytes. These observations and the magnitude of the proliferative response suggest that activation is polyclonal and involves mechanisms other than BLV antigen-specific stimulation.