The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G2 phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G2 arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G1 arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G2 arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G2 arrest. G2 arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G2 arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer

Genetic diversity of BoLA-DRB3 in South American Zebu cattle populations

Background: Bovine leukocyte antigens (BoLAs) are used extensively as markers of disease and immunological traits in cattle. However, until now, characterization of BoLA gene polymorphisms in Zebu breeds using high resolution typing methods has been poor. Here, we used a polymerase chain reaction sequence-based typing (PCR-SBT) method to sequence exon 2 of the BoLA class II DRB3 gene from 421 cattle (116 Bolivian Nellore, 110 Bolivian Gir, and 195 Peruvian Nellore-Brahman). Data from 1416 Taurine and Zebu samples were also included in the analysis. Results: We identified 46 previously reported alleles and no novel variants. Of note, 1/3 of the alleles were detected only in Zebu cattle. Comparison of the degree of genetic variability at the population and sequence levels with genetic distance in the three above mentioned breeds and nine previously reported breeds revealed that Zebu breeds had a gene diversity score higher than 0.86, a nucleotide diversity score higher than 0.06, and a mean number of pairwise differences greater than 16, being similar to those estimated for other cattle breeds. A neutrality test revealed that only Nellore-Brahman cattle showed the even gene frequency distribution expected under a balanced selection scenario. The FST index and the exact G test showed significant differences across all cattle populations (FST =0.057; p < 0.001). Neighbor-joining trees and principal component analysis identified two major clusters: one comprising mainly European Taurine breeds and a second comprising Zebu breeds. This is consistent with the historical and geographical origin of these breeds. Some of these differences may be explained by variation of amino acid motifs at antigen-binding sites. Conclusions: The results presented herein show that the historical divergence between Taurine and Zebu cattle breeds is a result of origin, selection, and adaptation events, which would explain the observed differences in BoLA-DRB3 gene diversity between the two major bovine types. This allelic information will be important for investigating the relationship between the major histocompatibility complex and disease, and contribute to an ongoing effort to catalog bovine MHC allele frequencies according to breed and location.Instituto de Genética Veterinari

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>DRA </it>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.</p> <p>Conclusions</p> <p>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.</p

Visualizing bovine leukemia virus (BLV)-infected cells and measuring BLV proviral loads in the milk of BLV seropositive dams

International audienceAbstractBovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission

BoLA-DRB3 genetic diversity in Highland Creole cattle from Bolivia

The genetic diversity of the BoLA‐DRB3 gene has been reported in different cattle breeds owing to its central role in the immune response. However, it is still unknown in hundreds of cattle breeds, especially native populations. Here, we studied BoLA‐DRB3 genetic diversity in Highland Creole cattle (CrAl) from Western Bolivia, raised at altitudes between 3800 and 4200 m. DNAs from 48 CrAl cattle were genotyped for BoLA‐DRB3 exon 2 alleles using polymerase chain reaction‐sequence‐based typing (PCR‐SBT). The results were compared with 1341 previously reported data from Tropical Creole cattle and other breeds raised in the region. Twenty‐three BoLA‐DRB3 alleles were identified in CrAl, including the BoLA‐DRB3*029:02 variant previously detected in other Creole cattle. Observed and expected heterozygosity were 0.87 and 0.93, respectively. Nucleotide diversity and the number of pairwise difference values were 0.078 and 19.46, respectively. The average number of nonsynonymous and synonymous substitutions were 0.037 and 0.097 for the entire BoLA‐DRB3 exon 2, and 0.129 and 0.388 for the antigen‐binding site, respectively. Venn analysis and the review of the IPD‐MHC database and the literature showed that 2 of 64 alleles were only detected in CrAl, including BoLA‐DRB3*029:01 previously reported in African cattle and *048:01 detected in Philippine cattle. Two additional alleles, BoLA‐DRB3*007:02 and *029:02, were only present in CrAl and Lowland Creole cattle. Principal Component Analysis (PCA) showed that Bolivian Creole cattle breeds were closely located but they were distant from the Colombian Hartón del Valle Creole. FST analysis showed a low degree of genetic differentiation between Highland and Lowland Bolivian Creole cattle (FST = 0.015). The present results contribute to increasing our knowledge of BoLA‐DRB3 genetic diversity in cattle breeds.Fil: Giovambattista, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Takeshima, Shin Nosuke. Jumonji University; JapónFil: Moe, Kyaw Kyaw. University of Veterinary Science; BirmaniaFil: Pereira Rico, Juan A.. Universidad Autonoma Gabriel Rene Moreno; BoliviaFil: Polat, Meripet. Viral Infectious Diseases Unit; JapónFil: Loza Vega, Ariel. Universidad Autonoma Gabriel Rene Moreno; BoliviaFil: Arce Cabrera, Orlando N.. Universidad Técnica de Oruro; BoliviaFil: Aida, Yoko. Viral Infectious Diseases Unit; Japó

A new genotype of bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis

Background: Bovine leukemia virus (BLV) is a member of retroviridae family, together with human T cell leukemia virus types 1 and 2 (HTLV-1 and -2) belonging to the genes deltaretrovirus, and infects cattle worldwide. Previous studies have classified the env sequences of BLV provirus from different geographic locations into eight genetic groups. To investigate the genetic variability of BLV in South America, we performed phylogenetic analyses of whole genome and partial env gp51 sequences of BLV strains isolated from Peru, Paraguay and Bolivia, for which no the molecular characteristics of BLV have previously been published, and discovered a novel BLV genotype, genotype-9, in Bolivia. Results: In Peru and Paraguay, 42.3 % (139/328) and over 50 % (76/139) of samples, respectively, were BLV positive. In Bolivia, the BLV infection rate was up to 30 % (156/507) at the individual level. In Argentina, 325/420 samples were BLV positive, with a BLV prevalence of 77.4 % at the individual level and up to 90.9 % at herd level. By contrast, relatively few BLV positive samples were detected in Chile, with a maximum of 29.1 % BLV infection at the individual level. We performed phylogenetic analyses using two different approaches, maximum likelihood (ML) tree and Bayesian inference, using 35 distinct partial env gp51 sequences from BLV strains isolated from Peru, Paraguay, and Bolivia, and 74 known BLV strains, representing eight different BLV genotypes from various geographical locations worldwide. The results indicated that Peruvian and Paraguayan BLV strains were grouped into genotypes-1, -2, and -6, while those from Bolivia were clustered into genotypes-1, -2, and -6, and a new genotype, genotype-9. Interestingly, these results were confirmed using ML phylogenetic analysis of whole genome sequences obtained by next generation sequencing of 25 BLV strains, assigned to four different genotypes (genotypes-1, -2, -6, and -9) from Peru, Paraguay, and Bolivia. Comparative analyses of complete genome sequences clearly showed some specific substitutions, in both structural and non-structural BLV genes, distinguishing the novel genotype-9 from known genotypes. Conclusions: Our results demonstrate widespread BLV infection in South American cattle and the existence of a new BLV genotype-9 in Bolivia. We conclude that at least seven BLV genotypes (genotypes-1, -2, -4, -5, -6, -7, and -9) are circulating in South America.Facultad de Ciencias VeterinariasInstituto de Genética Veterinari

Epidemiology and genetic diversity of bovine leukemia virus

Abstract Bovine leukemia virus (BLV), an oncogenic member of the Deltaretrovirus genus, is closely related to human T-cell leukemia virus (HTLV-I and II). BLV infects cattle worldwide and causes important economic losses. In this review, we provide a summary of available information about commonly used diagnostic approaches for the detection of BLV infection, including both serological and viral genome-based methods. We also outline genotyping methods used for the phylogenetic analysis of BLV, including PCR restriction length polymorphism and modern DNA sequencing-based methods. In addition, detailed epidemiological information on the prevalence of BLV in cattle worldwide is presented. Finally, we summarize the various BLV genotypes identified by the phylogenetic analyses of the whole genome and env gp51 sequences of BLV strains in different countries and discuss the distribution of BLV genotypes worldwide