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See detailMassive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites During Primary Infection
Gillet, Nicolas ULg; Gutiérrez, Gerónimo; Rodriguez, Sabrina ULg et al

in PLoS Pathogens (2013), 9(10),

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection generally asymptomatic but can also lead to leukemia or lymphoma. These ... [more ▼]

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection 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. [less ▲]

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See detailSafety of long-term treatment of HAM/TSP patients with valproic acid
Olindo, Stéphane; Belrose, Gildas; Gillet, Nicolas ULg et al

in Blood (2011), 118(24), 6306-6309

HTLV-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a neurodegenerative disease of the central nervous system induced by Human T lymphotropic virus type 1 (HTLV-1). As a potential ... [more ▼]

HTLV-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a neurodegenerative disease of the central nervous system induced by Human T lymphotropic virus type 1 (HTLV-1). As a potential therapeutic approach, we previously suggested reducing the proviral load (PVL) by modulating lysine deacetylase activity using valproic acid (VPA) and exposing virus-positive cells to the host immune response. We conducted a single-center, two-year open-label trial, with 19 HAM/TSP volunteers treated with oral VPA. PVL, CD38/HLA-DR expression and CD8+ lysis efficiency were not significantly affected by VPA. Mean scores of HAM/TSP disability did not differ between baseline and final visit. Walking Time Test (WTT) increased significantly (>20%) in 3 patients and was in keeping with minor VPA side effects (drowsiness and tremor). WTT improved rapidly after VPA discontinuation. We conclude that long term treatment with VPA is safe in HAM/TSP. [less ▲]

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See detailEvidencia de determinantes de patogenia en las regiones Long Terminal Repeats (LTRs) del genoma del virus de la leucemia bovina (BLV)
Rodriguez, Sabrina ULg; Trono, K.; Jones, L.R.

Poster (2011, September 26)

Evidencia de determinantes de patogenia en las regiones Long Terminal Repeats (LTRs) del genoma del virus de la leucemia bovina (BLV) Sabrina M. Rodríguez1*, Karina Trono2, Leandro R. Jones3 1 Molecular ... [more ▼]

Evidencia de determinantes de patogenia en las regiones Long Terminal Repeats (LTRs) del genoma del virus de la leucemia bovina (BLV) Sabrina M. Rodríguez1*, Karina Trono2, Leandro R. Jones3 1 Molecular and Cellular Epigenetics, Interdisciplinary Cluster for Applied Genoproteomics (GIGA), University of Liège (ULg), Belgium. 2 Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria INTA-Castelar, CC 25 (1712), Castelar. 3 División de Biología Molecular, Estación de Fotobiología Playa Unión, CC 15, Rawson, Chubut 9103, Argentina. *E-mail: sabrina.rodriguez@ulg.ac.be El resultado de la infección por el virus de la leucemia bovina (BLV) es diverso. La mayoría de los animales infectados resultan portadores asintomáticos (AL) y cerca del 30% desarrolla una condición benigna denominada linfocitosis persistente (LP). La forma tumoral o linfosarcoma (LS) afecta a un 1-5% de los bovinos infectados. Las bases genético-moleculares del desarrollo de las distintas formas clínicas son desconocidas. Las Repeticiones Terminales Largas (Long Terminal Repeats, LTR) del genoma viral constituyen determinantes genéticos de patogenia en el caso de otros retrovirus. Sin embargo, esta posibilidad no ha sido evaluada para el BLV. Los análisis para probar la correlación entre los caracteres clínicos y genotípicos entre especies deben ser corregidos incluyendo la filogenia del grupo. De otra manera, la historia evolutiva compartida puede comprometer la independencia estadística del análisis. Sobre estas bases, el objetivo de este trabajo consistió en estudiar la influencia de las variaciones genéticas de las regiones regulatorias LTR del BLV en el desarrollo de las diferentes formas clínicas de la infección mediante métodos comparativos filogenéticos, cladísticos y probabilísticos. Con este fin, se secuenció la región 5´LTR de 40 provirus obtenidos a partir de bovinos naturalmente infectados con BLV que presentaron las diferentes formas clínicas (AL, PL, LS). Fueron identificadas siete posiciones polimórficas que mostraron una asociación aparente con la presentación clínica. Una reconstrucción de la filogenia del grupo fue realizada a partir de las secuencias de la región env obtenidas para 28 de los 40 provirus estudiados en este trabajo. En conjunto, los análisis comparativos cladísticos y probabilísticos basados en el alineamiento empírico de las secuencias y la filogenia del grupo sugieren que las posiciones 41 y 56 del 5´LTR podrían estar correlacionadas con la presentación clínica. Los análisis probabilísticos indicaron además una asociación con la patogénesis viral para las posiciones 373, 450, 494 y 505, aunque los soportes estadísticos correspondientes fueron menores en comparación con los soportes obtenidos para las posiciones 41 y 56. Estas observaciones indican que las regiones LTR del BLV podrían constituir determinantes de patogenia. [less ▲]

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See detailPreventive and Therapeutic Strategies for Bovine Leukemia Virus: Lessons for HTLV
Rodriguez, Sabrina ULg; Florins, Arnaud-Francois ULg; Gillet, Nicolas ULg et al

in Viruses (2011), 3(7), 1210-1248

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of ... [more ▼]

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle, segregation of BLV-free animals and vaccination. Although having been instrumental in regions such as the EU, these strategies were unsuccessful elsewhere mainly due to economic costs, management restrictions and lack of an efficient vaccine. This review, which summarizes the different attempts previously developed to decrease seroprevalence of BLV, may be informative for management of HTLV-1 infection. We also propose a new approach based on competitive infection with virus deletants aiming at reducing proviral loads. [less ▲]

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See detailPathways of Human T-Lymphotropic Virus type I for viral entry
Boulanger, F.; Rodriguez, Sabrina ULg; Willems, L.

Scientific conference (2011, June 06)

Pathways of Human T-Lymphotropic Virus type I for viral entry Boulanger Fanny N.J., Rodriguez Sabrina and Willems Luc Cellular and Molecular Epigenetics, GIGA-Research, Liège The Human T-Lymphotropic ... [more ▼]

Pathways of Human T-Lymphotropic Virus type I for viral entry Boulanger Fanny N.J., Rodriguez Sabrina and Willems Luc Cellular and Molecular Epigenetics, GIGA-Research, Liège The Human T-Lymphotropic Virus type I (HTLV-1), the first discovered human retrovirus, infects an estimated number of 20 million people worldwide. HTLV-1 is the causative agent of Adult T-cell Leukemia/Lymphoma (ATL) and a neurodegenerative disease called HTLV associated myeolopathy / tropical spastic paraparesis (HAM/TSP). We have been interested in the early steps of HTLV entry into cells. Infection requires interaction of the viral envelope glycoproteins (SU and TM) with the cell membrane. Three molecules are required for binding and entry into cells: heparan-sulfate proteoglycans (HSPGs), the VEGF-165 receptor Neuropilin 1 (NRP-1) and the ubiquitous glucose transporter GLUT-1. Consecutive steps of HTLV-1 entry are poorly characterized. These could involve phagocytosis/macropinocytosis, caveolae pathway, clathrin-mediated endocytosis, and several mechanisms independent of these two organelles but mediated by dynamin. To identify the endocytic pathways of HTLV-1 entry, we used two complementary strategies based either on pharmacological inhibition or on dominant-negative proteins expression. First, we evaluated the effect of a series of pharmacological inhibitors to interfere with main endocytic pathways. We used wortmannin and cytochalasin D to interfere with phagocytosis and macropinocytosis through their action on phosphatidyl-inositol-3 kinase and on the actin network, respectively. Clathrin-mediated endocytosis was blocked with chlorpromazine and with a polyclonal anti-clathrin antibody as a direct competitor for the formation of clathrin-coated pits. The caveolar function was disrupted with the sterol-binding drug nystatin and the protein tyrosine-kinase inhibitor genistein. Finally, dynamin was competed with a peptide (Dynamin Inhibitory Peptide) or inhibited by dynasore. Since pharmacological inhibitors may have off-target effects, we also used an alternate approach based on dominant-negative proteins expressed by lentivirus-derived vectors. An AP-2-binding site from Eps15 (Epidermal Growth Factor Receptor Substrate 15) was used to interfere with clathrin-mediated endocytosis. A N-terminally GFP-tagged caveolin-1 construct acted as an inhibitor of the caveolae pathway. A dominant-negative GTP-binding mutant of dynamin2(aa) was used to interfere with dynamin-dependant internalization pathways. Lentiviruses expressing these dominant-negative proteins were transduced into several cell lines or primary cells (T-lymphocytes and dendritic cells). These transduced cells were then infected by co-cultivation with HTLV-1-infected T-lymphocytes (MT2) or with free particles. These different approaches will thus allow identifying the pathways involved in cell entry by HTLV-1. [less ▲]

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See detailA life-attenuated BLV deletant as a candidate vaccine to inhibit viral transmission in bovine herds
Rodriguez, Sabrina ULg; Gutiérrez, G.; Florins, A. et al

Scientific conference (2011, June 06)

A life-attenuated BLV deletant as a candidate vaccine to inhibit viral transmission in bovine herds Sabrina M. Rodríguez1*†, Gerónimo Gutiérrez2*, Arnaud Florins3, Lucas Vagnoni2, Irene Alvarez2, Nicolas ... [more ▼]

A life-attenuated BLV deletant as a candidate vaccine to inhibit viral transmission in bovine herds Sabrina M. Rodríguez1*†, Gerónimo Gutiérrez2*, Arnaud Florins3, Lucas Vagnoni2, Irene Alvarez2, Nicolas Gillet1, Karina Trono2‡, Luc Willems1,3‡ *‡S.M. Rodríguez / G. Gutiérrez and K.Trono / L. Willems contributed equally to this work. 1 Molecular and Cellular Epigenetics, Interdisciplinary Cluster for Applied Genoproteomics (GIGA), University of Liège (ULg), Liège (4100), Belgium. 2 Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria, (1712), Castelar, Argentina. 3 Molecular and Cellular Biology, Gembloux Agro-Bio Tech, University of Liège (ULg), Gembloux (5030), Belgium †E-mail: sabrina.rodriguez@ulg.ac.be Bovine Leukemia Virus (BLV) is a major sanitary concern in many countries where the virus is widely disseminated among dairy herds with obvious economic impact. Different control strategies have been implemented worldwide to control BLV infection or eradicate the disease with diverse success. Eradication by culling is not economically sustainable in highly infected regions such as Argentina, US or Japan. Segregation of BLV-infected cattle is expensive due to duplication of facilities. Finally, several candidate vaccines based on recombinant viral proteins were unsuccessful to protect from challenge. Therefore, here we propose a novel strategy aimed to decrease seroprevalence based on the employ of a life-attenuated BLV provirus as a candidate vaccine. The rationale behind this strategy is the deletion of genes required to induce pathogenesis leaving those involved in infectivity, resulting in an attenuated deletant with impaired transmissibility. Preliminary experiments showed that the deletant provirus is infectious and elicits an efficient immune response in sheep (n=3) and in the natural host, bovines (n=9). Lack of spread to sentinels further supports the safety of the vaccine. Based on these promissory results, an ongoing validation program is being performed to evaluate the capacity of the candidate vaccine to protect from wild-type BLV infection in herd conditions (n=105). Infection will be routinely monitored and proviral loads will be determined. The efficiency of the immune response will be evaluated by titration of specific antibodies, cytotoxic lysis efficiency and cytokine profile. Viral expression ex vivo and provirus clonality will be also evaluated. This data will be instrumental for understanding the basic mechanisms undergoing during BLV infection and for elaborating a novel vaccine. We do believe this practical and cost-effective vaccination strategy is the sole economically viable in countries with high prevalence. [less ▲]

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See detailHTLV-1 clonality during chronic infection and BLV clonality during primary infection
Gillet, N.; Hlela, C.; Verdonck, T. et al

in AIDS Research and Human Retroviruses (2011, June 06), 8(1), 185

HTLV-1 clonality during chronic infection and BLV clonality during primary infection Nicolas A Gillet1,2*, Carol Hlela1, Tine Verdonck3, Eduardo Gotuzzo3, Daniel Clark3, Sabrina Rodriguez2, Nirav Malani4 ... [more ▼]

HTLV-1 clonality during chronic infection and BLV clonality during primary infection Nicolas A Gillet1,2*, Carol Hlela1, Tine Verdonck3, Eduardo Gotuzzo3, Daniel Clark3, Sabrina Rodriguez2, Nirav Malani4, Anat Melamed1, Niall Gormley5, Richard Carter5, David Bentley5, Charles Berry6, Frederic D Bushman4, Graham P Taylor7, Luc Willems2, Charles R M Bangham1 1Department of Immunology, Wright-Fleming Institute, Imperial College London, London, W2 1PG, UK. 2Molecular and Cellular Epigenetics, Interdisciplinary Cluster for Applied Genoproteomics (GIGA) of University of Liège (ULg), Liège, 4000, Belgium. 3Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru. 4Department of Microbiology, University of Pennsylvania School of Medicine, Pennsylvania, Philadelphia, PA, 19104, USA. 5Illumina, Chesterford Research Park, Essex, Little Chesterford, CB10 1XL, UK. 6University of California, California, La Jolla San Diego, CA, 92093-0901, USA. 7Department of Genitourinary Medicine and Communicable Diseases, Wright-Fleming Institute, Imperial College London, London, W2 1PG, UK. HTLV-1 persists by driving clonal proliferation of infected T-lymphocytes. A high proviral load predisposes to the inflammatory and malignant diseases associated with HTLV-1. Yet the reasons for the remarkable variation within and between individuals in the abundance of HTLV-1-infected clones remain unknown. We demonstrate that negative selection dominates during chronic infection, favouring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We postulated that this selection occurred mainly during the primary infection. We are testing this hypothesis in an animal model by studying the BLV clonality during the primary infection in cows. By measuring the proviral load, the anti-BLV immune response and the BLV clonality we aim to quantify the impact of the immune response on the rate of infectious spread and on the selection of proviruses inserted in a particular genomic environment. Co-infection with Strongyloides stercoralis or Staphylococcus appears to be another risk factor for the development of HTLV-1 associated diseases. We observed that HTLV-1 clonality is altered by co-infection with these pathogens with an increase of both the number and the abundance of the infected T-cell clones. The genomic characteristics of the proviral integration sites in the most abundant clones differ significantly between co-infected individuals and those with HTLV-1 alone, implying the existence of different selection forces in co-infected patients. The rate of appearance of new clones in patients co-infected with Strongyloides stercoralis is higher than in patients with HTLV-1 alone. By comparing skin lesions and blood samples from patients with Infective Dermatitis associated with HTLV-1 (IDH), we observed a significant proportion of distinct infected clones between the two compartments. The skin lesions seem to be a site for HTLV-1 infectious spread. [less ▲]

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See detailA life-attenuated BLV deletant as a candidate vaccine to inhibit viral transmission in bovine herds
Gutiérrez, G.; Rodriguez, Sabrina ULg; Florins, A. et al

in AIDS Research and Human Retroviruses (2011, June 05), 8(1), 12

A life-attenuated BLV deletant as a candidate vaccine to inhibit viral transmission in bovine herds Gerónimo Gutiérrez2*, Sabrina M. Rodríguez1*†, Arnaud Florins3, Lucas Vagnoni2, Irene Alvarez2, Nicolas ... [more ▼]

A life-attenuated BLV deletant as a candidate vaccine to inhibit viral transmission in bovine herds Gerónimo Gutiérrez2*, Sabrina M. Rodríguez1*†, Arnaud Florins3, Lucas Vagnoni2, Irene Alvarez2, Nicolas Gillet3, Karina Trono2‡, Luc Willems1,3‡ *‡S.M. Rodríguez / G. Gutiérrez and K.Trono / L. Willems contributed equally to this work. 1 Molecular and Cellular Epigenetics, Interdisciplinary Cluster for Applied Genoproteomics (GIGA), University of Liège (ULg), Liège (4100), Belgium. 2 Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria, (1712), Castelar, Argentina. 3 Molecular and Cellular Biology, Gembloux Agro-Bio Tech, University of Liège (ULg), Gembloux (5030), Belgium †E-mail: sabrina.rodriguez@ulg.ac.be There are different strategies to reduce BLV prevalence. Eradication by culling of infected animals is not economically sustainable in highly infected regions such as Argentina, US or Japan. Partition and confinement on BLV-infected cows is expensive due to duplication of facilities. Finally, several candidate vaccines based on recombinant viral proteins were unsuccessful to protect from challenge. Facing these failures, we propose a novel strategy based on the use of a live-attenuated BLV provirus. The rationale behind this strategy relies on the deletion of genes required to induce pathogenesis leaving those involved in infectivity, resulting in an attenuated mutant with impaired transmissibility. In a first set of experiments, we show that the mutant is infectious and elicits an efficient immune response in sheep (n=3) and in cows (n=9). Lack of spread to uninfected sentinels further supports the safety of the vaccine. Based on these promising results, a validation program in herd (n=105) is ongoing to evaluate the capacity of the candidate vaccine to protect from wild-type BLV infection. The following experiments are carried out: quantification of the proviral loads, assessment of immune response efficiency (antibody titers, CTL response and cytokine profiling), measure of viral expression in vivo (qRT-PCR) and ex vivo (expression of Tax and p24gag) and determination of provirus clonality during infection. This data will be instrumental for understanding the basic mechanisms undergoing during BLV infection and for elaborating of a novel vaccine. [less ▲]

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See detailEvidence for the existence of pathogenicity determinants in the Long Terminal Repeats (LTRs) of the Bovine Leukemia Virus (BLV) genome
Rodriguez, Sabrina ULg; Trono, K.; Jones, L.R.

in AIDS Research and Human Retroviruses (2011), 8(1), 26

Evidence for the existence of pathogenicity determinants in the Long Terminal Repeats (LTRs) of the Bovine Leukemia Virus (BLV) genome Sabrina M. Rodríguez1*, Karina Trono2, Leandro R. Jones3 1 Molecular ... [more ▼]

Evidence for the existence of pathogenicity determinants in the Long Terminal Repeats (LTRs) of the Bovine Leukemia Virus (BLV) genome Sabrina M. Rodríguez1*, Karina Trono2, Leandro R. Jones3 1 Molecular and Cellular Epigenetics, Interdisciplinary Cluster for Applied Genoproteomics (GIGA) , University of Liège (ULg), Belgium. 2 Instituto de Virología, CICVyA, Instituto Nacional de Tecnología Agropecuaria INTA-Castelar, CC 25 (1712), Castelar. 3 División de Biología Molecular, Estación de Fotobiología Playa Unión, CC 15, Rawson, Chubut 9103, Argentina. *E-mail: sabrina.rodriguez@ulg.ac.be The majority of BLV-infected animals are asymptomatic carriers (AL) while about 30% develop a benign persistent lymphocytosis (PL). Fatal lymphosarcoma (LS) occurs in 5% of infected animals. The genetic basis of these diverse outcomes of BLV infection is still unknown. Viral LTRs constitute a genetic determinant of pathogenesis for other retroviruses. However, this possibility has never been tested for BLV. Analyses to test correlation between clinical and genotypic traits across species must be corrected by including the group phylogeny. Otherwise, shared evolutionary history can jeopardize statistical independence. Thus, the influence of BLV LTR genetic variation on the clinical manifestation of the disease was investigated by employing Cladistic and Probabilistic, phylogenetic comparative methods. With this purpose, the 5´LTR region of 40 BLV proviruses from bovines with different clinical presentations (AL, PL, LS) was sequenced. Seven polymorphic positions showing an apparent association with the clinical presentation were identified. A provirus phylogeny was obtained using env gene sequences from 28 of the 40 provirus studied in this work. Both Cladistic and Probabilistic comparative analyses based on the empirical sequence alignment and the provirus phylogeny suggested that positions 41 and 56 might be correlated to the clinical presentation. The probabilistic analysis further indicated an association with the viral pathogenesis for positions 373, 450, 494 and 505, though the corresponding statistical supports were lower in comparison to the supports obtained for positions 41 and 56. These observations indicate that the BLV LTRs might contain pathogenicity determinants. [less ▲]

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See detailThe impact of HAART initiation on the selection and persistence of HIV-1 CTL-escape mutations
Dilernia, D.A.; Monaco, D.C.; Jones, L.R. et al

in AIDS Research and Human Retroviruses (2010, October), 26(10), 72

The impact of HAART initiation on the selection and persistence of HIV-1 CTL-escape mutations D.A. Dilernia,1 D.C. Monaco,1 L.R. Jones,2 G.D. Damilano,1 S. Rodriguez,3 H. Salomon1 1Argentinean Reference ... [more ▼]

The impact of HAART initiation on the selection and persistence of HIV-1 CTL-escape mutations D.A. Dilernia,1 D.C. Monaco,1 L.R. Jones,2 G.D. Damilano,1 S. Rodriguez,3 H. Salomon1 1Argentinean Reference Center for AIDS, Capital Federal, Argentina; 2División de Biología Molecular, Estación de Fotobiología, Playa Unión, Rawson, Chubut, Argentina; 3INTA, Castelar, Buenos Aires, Argentina Background: Immune response drives the selection of CTL escape mutations during the course of HIV infection. After initiation of HAART, antiviral drugs exert a strong selective force leading to an important limitation of viral replication. Our objective was to evaluate the impact of HAART initiation on the selection and persistence of CTL-escape mutations. Methods: Blood samples were collected from 113 newly HIV diagnosed individuals. A second sample was collected from 49 of them after 3 years. (12 of which were still drug-naı¨ve in the second sample). Dynamics of CTL-escape mutations identified in the gag gene by statistical analysis of HLA genes and viral sequences, including multiple comparison corrections (BH method) and phylogeny correction (Bayesian MCM method), were analyzed. Epitope-specific immune response was evaluated by ELISPOT against sequence-based designed peptides. Results: Immune response was evaluated on 113 individuals on an HLA-allele basis. Positive responses were detected against 6 of the 9 epitopes containing HLA-associated CTL-escape mutations.A03-restricted epitope RLRPGGKKK had the highest frequency of detection in allele-matched individuals (75%). Epitopes harboring the CTL-escape mutations identified through negative associations (i.e. escape is consensus) had lower frequency of detection: A02-restricted SLYNTVATL(25%) and A24-resctricted KYKLKHIVW(0%). Epitope sequences analysis over the second sample of 21 patients successfully sequenced showed that 38% of them did not have evidence of escape neither in the first sample nor in the second, 44% had the escape mutations in both samples and in 18%the escape mutations appeared in the second sample. Of the 7 individuals containing the epitopes of the last group, only 2 had initiated HAART (28.6%) while 64.3% (9/14) of individuals where no escape emerged, had initiated HAART. Conclusion: Accumulation of CTL-escape mutations at the population-level impaired recognition by individuals studied while initiation of HAART may prevent the selection of new escape mutations even in advanced stages of infection. [less ▲]

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See detailBovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel clades.
Rodriguez, Sabrina ULg; Golemba, Marcelo D.; Campos, Rodolfo H. et al

in Journal of General Virology (The) (2009), 90(Pt 11), 2788-97

Previous studies have classified the env sequences of bovine leukemia virus (BLV) provirus from different locations worldwide into between two and four genetic groupings. These different studies gave ... [more ▼]

Previous studies have classified the env sequences of bovine leukemia virus (BLV) provirus from different locations worldwide into between two and four genetic groupings. These different studies gave unique names to the identified groups and no study has yet integrated all the available sequences. Thus, we hypothesized that many of the different groups previously identified actually correspond to a limited group of genotypes that are unevenly distributed worldwide. To examine this hypothesis, we sequenced the env gene from 28 BLV field strains and compared these sequences to 46 env sequences that represent all the genetic groupings already identified. By using phylogenetic analyses, we recovered six clades, or genotypes, that we have called genotypes 1, 2, 3, 4, 5 and 6. Genotypes 1-5 have counterparts among the sequence groupings identified previously. One env sequence did not cluster with any of the others and was highly divergent when compared with the six genotypes identified here. Thus, an extra genotype, which we named 7, may exist. Similarity comparisons were highly congruent with phylogenetic analyses. Furthermore, our analyses confirmed the existence of geographical clusters. [less ▲]

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See detailLong-term treatment with valproic acid does not alleviate the condition of HAM/TSP
Olindo, S.; Belrose, G.; Lezin, A. et al

in AIDS Research and Human Retroviruses (2009, July 01), 25(11), 21

Long-Term Treatment with Valproic Acid Does Not Alleviate the Condition of HAM/TSP Olindo, S.,1 Belrose, G.,2 Lezin, A.,2 Gillet, N.,3 Defoiche, J.,4 Rodriguez, S.,4 Signaté, A.,1 Verlaeten, O.,4 Smadja ... [more ▼]

Long-Term Treatment with Valproic Acid Does Not Alleviate the Condition of HAM/TSP Olindo, S.,1 Belrose, G.,2 Lezin, A.,2 Gillet, N.,3 Defoiche, J.,4 Rodriguez, S.,4 Signaté, A.,1 Verlaeten, O.,4 Smadja, D.,1 Césaire, R.,2 Willems, L.4 1Service de Neurologie and JE 2503, Centre Hospitalier Universitaire de Fort-de-France, 97200 Fort-de-France, Martinique, France; 2Laboratoire de Virologie-Immunologie and JE 2503, Centre Hospitalier Universitaire de Fort-de-France, 97200 Fort-de-France, Martinique, France; 3Department of Immunology, Imperial College, London, UK; 4Molecular and Cellular Biology, University Academia ‘‘Wallonie-Europe’’, 5030 Gembloux, Belgium. LW is Research Director of the FNRS. We previously proposed to interfere with proviral loads in HAM/TSP patients by modulating lysine deacetylase activity using valproic acid (VPA). The strategy aims at activating viral gene expression in order to expose virus-positive cells to the host immune response. We conducted a single-center, two-year open-label trial, with 19 HAM/TSP volunteers treated with oral doses of VPA (20mg/Kg/day). Objectives were to assess biological response and clinical safety to VPA treatment in HAM/TSP patients. By microarray analysis, we show that VPA treatment moderately stimulated expression of cyclinD2 and Rho GTPase activating protein 18 in CD4-T cells. CD8-mediated lysis efficiency of Tax-expressing cells was unaltered by VPA treatment. The CD4-T cell turnover rate was calculated by GC/MS analysis from quantitative incorporation of deuterium into DNA. Transient increase in proviral loads correlated with accelerated proliferation. After 2 years, the proviral loads reached levels similar to those before treatment. The main clinical side effects were drowsiness (52%), tremor (47%), digestive symptoms (37%), vertigo (26%), and alopecia (10%). The frequency of side symptoms tended to decrease over the trial course. The neurological status over the study constituted the primary clinical safety measures. Disability Status Scale, muscle testing score, Ashworth score, and urinary dysfunction score showed no significant changes. Walking Time Test (WTT) slightly varied over the study except in 3 patients in whom the WTT variation rates were>20%. These 3 patients experienced drowsiness and tremor and improved rapidly after treatment discontinuation. Together, these observations indicated that long term treatment with VPA is safe but does not alleviate the condition of HAM/TSP. [less ▲]

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See detailBovine Leukemia Virus (BLV) could be subdivided into up to seven groups based on phylogenetic and similarity analyses of sequences from the env gene
Rodriguez, Sabrina ULg; Golemba, M.D.; Campos, R. et al

in AIDS Research and Human Retroviruses (2009, July 01), 25(11), 16

Bovine Leukemia Virus (BLV) Could be Subdivided into up to Seven Groups Based on Phylogenetic and Similarity Analyses of Sequences from the env Gene. Rodríguez, S.M., Trono, K., Jones, L.R., Golemba, M ... [more ▼]

Bovine Leukemia Virus (BLV) Could be Subdivided into up to Seven Groups Based on Phylogenetic and Similarity Analyses of Sequences from the env Gene. Rodríguez, S.M., Trono, K., Jones, L.R., Golemba, M., Campos, R. Instituto De Virología - Cicvya - Inta Castelar We sequenced the env gene from 28 BLV field strains from Argentina, and combined these sequences with 45 env sequences representing all the genetic groupings identified previously. These data were analyzed by phylogenetic analysis and similarity comparisons. Phylogenetic analyses showed the existence of five groups of sequences or genotypes. Four genotypes were supported by high bootstraps (89–100) and posterior probabilities (p=1.0), and the fifth one was supported by a high posterior probability (p=1.0) and moderate bootstrap (80–96) values. All these genotypes have one or more counterparts among the groupings identified previously, and thus many groups that were originally named differently correspond actually to the same genotype. Two sequences did not cluster with any other sequence, and were highly divergent when compared to the five groups mentioned above. This indicates that two extra genotypes could exist. The similarity comparisons were highly congruent with the phylogenetic analyses. The mean number of pair wise nucleotide substitutions between sequences from the same genotype was 11.1 (min. 6.5; max. 19), whereas it was 39.6 (min.36.8; max. 47.9) between sequences from different genotypes. Likewise, the mean number of amino acid substitutions in pair wise comparisons at the intra-genotype level was 3.3 (min. 0.4; max. 6.2), whereas it was 9.6 (min. 8.3; max. 13.5) at the inter-genotype level. Our analyses confirmed the existence of geographic clusters (p=6.838E-15, Chi2 test; p=4.583E-12, Fisher’s exact test). [less ▲]

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See detailDetection of bovine leukemia virus specific antibodies using recombinant p24-ELISA.
Gutierrez, G.; Alvarez, I.; Fondevila, N. et al

in Veterinary Microbiology (2009), 137(3-4), 224-34

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was ... [more ▼]

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was expressed in the Escherichia coli expression system, purified in a nickel charged resin and used as antigen in the ELISA test. A cut-off point was established considering the distribution of reactivity values obtained by rp24-ELISA on a set of 555 field serum samples that were stated as double positive (DP) or double negative (DN) by the combination of commercial agar gel immunodiffusion (AGID) assay and gp51-ELISA tests results. The rp24-ELISA showed good concordance with the agar gel immunodiffusion assay, when 710 serum samples were analyzed. In addition, rp24-ELISA demonstrated to be a precise assay with good repeatability and reproducibility and a better analytical sensitivity than AGID. From 67 discordant rp24-ELISA-AGID sera, 4 negative reactors were from the DP group, 28 positive samples were from the DN group and 35 positive samples were stated as negative by AGID but positive by the gp51-ELISA. Samples from this last subgroup were sent to an OIE reference laboratory where 28 samples were stated as negative, 5 samples were stated as positive and 2 as inconclusive. Complete concordance with blind previous results from an international proficiency test panel confirmed the capability of the assay to discriminate between infected and non-infected animals. In conclusion, the rp24-ELISA developed and standardized demonstrated to have good analytical characteristics to be considered for screening of BLV. [less ▲]

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See detailHLA-driven convergence of HIV-1 viral subtypes B and F toward the adaptation to immune responses in human populations.
Dilernia, Dario Alberto; Jones, Leandro; Rodriguez, Sabrina ULg et al

in PLoS ONE (2008), 3(10), 3429

BACKGROUND: Cytotoxic T-Lymphocyte (CTL) response drives the evolution of HIV-1 at a host-level by selecting HLA-restricted escape mutations. Dissecting the dynamics of these escape mutations at a ... [more ▼]

BACKGROUND: Cytotoxic T-Lymphocyte (CTL) response drives the evolution of HIV-1 at a host-level by selecting HLA-restricted escape mutations. Dissecting the dynamics of these escape mutations at a population-level would help to understand how HLA-mediated selection drives the evolution of HIV-1. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a study of the dynamics of HIV-1 CTL-escape mutations by analyzing through statistical approaches and phylogenetic methods the viral gene gag sequenced in plasma samples collected between the years 1987 and 2006 from 302 drug-naive HIV-positive patients. By applying logistic regression models and after performing correction for multiple test, we identified 22 potential CTL-escape mutations (p-value<0.05; q-value<0.2); 10 of these associations were confirmed in samples biologically independent by a Bayesian Markov Chain Monte-Carlo method. Analyzing their prevalence back in time we found that escape mutations that are the consensus residue in samples collected after 2003 have actually significantly increased in time in one of either B or F subtype until becoming the most frequent residue, while dominating the other viral subtype. Their estimated prevalence in the viral subtype they did not dominate was lower than 30% for the majority of samples collected at the end of the 80's. In addition, when screening the entire viral region, we found that the 75% of positions significantly changing in time (p<0.05) were located within known CTL epitopes. CONCLUSIONS: Across HIV Gag protein, the rise of polymorphisms from independent origin during the last twenty years of epidemic in our setting was related to an association with an HLA allele. The fact that these mutations accumulated in one of either B or F subtypes have also dominated the other subtype shows how this selection might be causing a convergence of viral subtypes to variants which are more likely to evade the immune response of the population where they circulate. [less ▲]

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See detailEvidence of HLA-mediated immune response driving the maintenance of CTL-escape variants in patients with undetectable viral load
Dilernia, D.; Lourtau, L.; Jones, L.R. et al

in AIDS Research and Human Retroviruses (2008, October 13), 4(1), 79

Evidence of HLA-mediated immune response driving the maintenance of CTL-escape variants in patients with undetectable viral load. Dario A Dilernia, L Lourtau, L Jones, S Rodriguez, C Bautista, M Gomez ... [more ▼]

Evidence of HLA-mediated immune response driving the maintenance of CTL-escape variants in patients with undetectable viral load. Dario A Dilernia, L Lourtau, L Jones, S Rodriguez, C Bautista, M Gomez-Carrillo, M Losso, and H Salomon. Immune response drives the selection of CTL-escape mutations during the course of HIV infection. After initiation of HAART, antiviral drugs exert a stronger selective force leading to a higher limitation of viral replication. Our objective was to evaluate the effectiveness of immune response as a selective force in a context of extremely reduced viral population size. Gag gene was sequenced and HLA-A and B genotyped over 108 samples from drug-naïve HIV-1 positive individuals. Associations between HLA alleles and viral polymorphisms were assessed by logistic regression. Multiple comparison corrections were addressed by the BH method (q-values). Phylogeny correction was performed by a Bayesian Markov-Chain Monte-Carlo method. Analysis of site-specific synonymous and non-synonymous substitution rate was assessed through the codon-based ML IFEL method. For four patients with viral load < 50 copies/ml (identified according to their HLA profile), RNA extraction was performed from 7 ml of plasma through an ultracentrifugation-based method and gag gene amplified through a Hi-Fi PCR. 20-40 clones were sequenced in samples obtained previously and during HAART. We found that HLA-B57 and A03 were the most efficient alleles in forcing CTL-escape, targeting mainly the previously characterized epitopes TSTLQEQIGW(p=0.0002) and RLRPGGKKK(p<10E-7), respectively. Sites under significant positive selection (p<0.05) during HAART were position 20 (within A03-restricted RLRPGGKK) for patient A02A3B35B39, position 385 (within A3-restricted RGNFRNQRK) for patient A3A31B7B45 and position 84 (within A2-restricted SLYNTVATL) for patient A2A3B39B57. Epitopes sequences of RLRPGGKKK and TSTLQEQIGWF were in the escape state for patients harboring the selective alleles. In spite of the low viral diversity achieved during HAART, we detected sites under positive selection. Our results show that during successful HAART, targeted CTL-epitopes are still forced to evolve adaptively, suggesting that CTL-mediated immune response would be able to keep driving the evolution of HIV variants even in viral population with a remarkable low replication rate. [less ▲]

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See detailDiversidad genética del Virus de la Leucemia Bovina: una aproximación filogenética
Rodriguez, Sabrina ULg; Trono, K.; Jones, L.R.

Poster (2008, September 22)

Diversidad genética del Virus de la Leucemia Bovina: una aproximación filogenética Sabrina M. Rodríguez1, Karina Trono1, Leandro R. Jones2 1 Instituto de Virología, CICVyA, INTA-Castelar. C.C. 77. C.P ... [more ▼]

Diversidad genética del Virus de la Leucemia Bovina: una aproximación filogenética Sabrina M. Rodríguez1, Karina Trono1, Leandro R. Jones2 1 Instituto de Virología, CICVyA, INTA-Castelar. C.C. 77. C.P. 1712. Castelar, Buenos Aires, Argentina. 2 Estación de Fotobiología Playa Unión. C.C. 15. C.P. 9103. Rawson, Chubut, Argentina. *E-mail: srodriguez@cnia.inta.gov.ar El virus de la leucemia bovina (BLV), agente etiológico de la Leucemia Bovina Enzoótica (LBE), pertenece al género Deltaretrovirus, dentro de la familia Retroviridae. En Argentina, la infección por BLV presenta niveles de seroprevalencia individual del 32,8% y del 84% a nivel predial. En trabajos anteriores, las cepas de BLV han sido clasificadas en dos grupos: uno Europeo y otro que incluye cepas de Japón y Estados Unidos (USA). En dos trabajos mas recientes se observo que la mayoría de las cepas argentinas se agrupan en un clado, mientras que una proporción minoritaria se agrupa con cepas de diverso origen. En uno de estos trabajos se propuso la existencia de cuatro genogrupos (Clusters I a IV), uno de los cuales (Cluster II) seria exclusivo de Argentina. Estas clasificaciones fueron propuestas en base a análisis realizados a partir de matrices de distancias obtenidas a partir de secuencias parciales (444/1547 pb.) del gen env. Las distancias fueron calculadas asumiendo el modelo evolutivo de Kimura con dos parámetros (K2P) y los árboles se obtuvieron mediante el algoritmo Neighbor-Joining (NJ). Actualmente, existen métodos específicos y objetivos para la selección de modelos evolutivos. Se ha demostrado que el uso de modelos arbitrarios puede conducir a resultados inesperados y erróneos. Por otro lado, el algoritmo NJ ha sido ampliamente superado por métodos más exhaustivos y modernos cómo Parsimonia y Máxima Verosimilitud. En este trabajo se caracterizaron 28 provirus argentinos mediante la secuenciación del gen env (1333/1547 pb.). Estas secuencias, junto con otras 42 secuencias seleccionadas de forma tal de abarcar toda la diversidad conocida y de incluir representantes de la mayor cantidad posible de locaciones geográficas, fueron analizadas mediante Parsimonia con el objeto de examinar las clasificaciones previas. Como outgroup (OG), se utilizaron secuencias homólogas de HTLV-1, HTLV-2, STLV-1 y STLV-2. Las secuencias fueron alineadas usando el programa Muscle 3.6. Los alineamientos fueron analizados con el programa TNT 1.1, mediante el cual se realizaron 1000 adiciones al azar (RAS) seguidas de Tree Bisection Reconection (TBR). La estimación del soporte de los grupos identificados se realizó mediante Symetric Jackniffing, utilizando el programa TNT. Nuestros resultados soportan la existencia del clado de secuencias de Japón y USA. Los árboles obtenidos en este trabajo no apoyan ni contradicen el grupo europeo, ya que el mismo podría estar presente en algunas de las posibles resoluciones del árbol sin raíz. Las secuencias de Argentina no se ubicaron en un único grupo sino que se mezclaron con cepas de diverso origen. Nuestros resultados no soportaron la hipótesis de que exista regionalización de las cepas de BLV, tal como se ha observado en HTLV. El árbol consenso de los árboles derivados del análisis incluyendo el outgroup, presentó una resolución relativamente baja con respecto al árbol consenso derivado del análisis sin raíz. Esto parece deberse a que las secuencias utilizadas como outgroup son extremadamente divergentes con respecto al ingroup, lo cual queda en evidencia luego de una simple inspección visual del correspondiente alineamiento. El resultado de esto es que muchas regiones de la matriz constituyen alineamientos al azar (es decir asignaciones de homología primaria incorrectas y aleatorias), con la consecuente pérdida de información. Esta última observación es de particular interés dado que HTLV y STLV han sido utilizados como OG en una gran cantidad de trabajos, lo cual está justificado ya que son los grupos conocidos más próximos a BLV. Nuestros análisis, sin embargo, sugieren fuertemente que es necesario identificar outgroups más adecuados para lograr una comprensión más acabada de la diversidad de estos virus. [less ▲]

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See detailEstudio Comparativo de la asociación entre variantes genotípicas del Virus de la Leucemia Bovina y la forma clínica de la infección
Rodriguez, Sabrina ULg; Trono, K.; Jones, L.R.

Poster (2008, September 22)

Estudio Comparativo de la asociación entre variantes genotípicas del Virus de la Leucemia Bovina y la forma clínica de la infección. Sabrina M. Rodríguez1*, Karina Trono1, Leandro R. Jones1,2. 1 Instituto ... [more ▼]

Estudio Comparativo de la asociación entre variantes genotípicas del Virus de la Leucemia Bovina y la forma clínica de la infección. Sabrina M. Rodríguez1*, Karina Trono1, Leandro R. Jones1,2. 1 Instituto de Virología, CICVyA, INTA-Castelar. C.C. 77. C.P. 1712. Castelar, Buenos Aires, Argentina. 2 Dirección actual: Estación de Fotobiología Playa Unión. C.C. 15. C.P. 9103. Rawson, Chubut, Argentina. *E-mail: srodriguez@cnia.inta.gov.ar Introducción. El Virus de la Leucemia Bovina (BLV) es un retrovirus oncogénico que infecta bovinos y es el agente causal de la Leucosis Bovina Enzoótica. El resultado de la infección por BLV es diverso. La mayoría de los individuos infectados (70%) resultan portadores asintomáticos o aleucémicos (AL) y cerca del 25% desarrolla una condición benigna denominada Linfocitosis Persistente (LP). La forma tumoral o linfosarcoma (LS), afecta a un 1-5% de los bovinos infectados que desarrollan una neoplasia B- linfoproliferativa. Las bases genético-moleculares del desarrollo de estas variadas presentaciones clínicas de la infección por BLV son hasta el momento desconocidas. Las secuencias provirales de Repeticiones Terminales Largas (Long Terminal Repeats, LTR) de los retrovirus han sido implicadas en la determinación del tropismo, la virulencia y la patogenicidad, jugando, además, un rol importante en la oncogénesis. Sin embargo, la influencia de la variación genética en las regiones LTR sobre aspectos clínicos de la infección por BLV no ha sido todavía estudiada. Los objetivos de este trabajo fueron caracterizar estas regiones regulatorias y analizar la posible asociación entre las variaciones genéticas del LTR de BLV y las formas clínicas de la infección (AL, PL, LS). Materiales y Métodos. Se amplificó y secuenció la región LTR de 40 provirus obtenidos a partir de bovinos naturalmente infectados por BLV que presentaron las diferentes formas clínicas de la infección. La relación entre caracteres genotípicos y fenotípicos para cada posición variable del LTR fue evaluada mediante métodos comparativos filogenéticos. Para ello se utilizó una filogenia obtenida a partir de secuencias de la región env. La reconstrucción de caracteres en árboles-espejo se llevó a cabo mediante el programa Mesquite y el posterior análisis estadístico de los datos fue realizado mediante el Test Exacto de Fisher. Asimismo, se empleó el Test de Correlación de Pagel implementado en el programa BayesTraits-Discrete para estudiar la existencia de correlación en un marco estadístico-filogenético. Resultados. El análisis de las secuencias de los LTRs reveló la presencia de 7 posiciones polimórficas que podrían variar correlacionadamente con las presentaciones clínicas de la infección (AL/LP y LS). Es decir que la presencia de ciertas bases en determinadas posiciones del LTR podría asociarse con la correspondiente manifestación patogénica asociada a la infección. Los análisis basados en árboles-espejo indicaron que en las posiciones 41 y 56 del LTR dicha asociación es estadísticamente significativa (p<0.05, test Exacto de Fisher). El análisis de Máxima Verosimilitud (modo ML del programa Bayes Traits Discrete) indicó que, para las 7 posiciones analizadas, los datos se ajustan mejor a un modelo en el cual la forma clínica se correlaciona con la genética viral. Resultados similares se observaron mediante la utilización de simulaciones de Monte Carlo (modo MCMC del programa Bayes Traits Discrete): en todas las posiciones los datos se ajustan mejor al modelo dependiente o de correlación (LR > 0). Sin embargo, los valores de LR para todas las posiciones estudiadas no son concluyentes, ya que los valores de LR fueron menores que 2. Conclusión. Los resultados reportados en este trabajo sugieren que al menos dos posiciones de la región LTR (41 y 56) podrían estar implicadas en la determinación de la evolución clínica de los animales infectados por BLV. Estos hallazgos podrían contribuir al conocimiento respecto de las bases moleculares de la patogénesis para este virus. [less ▲]

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See detailDinámica de la infección perinatal con Virus de la Leucosis Bovina en el rodeo lechero
Rossich, G.; Gutiérrez, G.; Rodriguez, Sabrina ULg et al

Poster (2008, September 22)

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See detailDinámica de la excreción natural del Virus de la Leucosis Bovina en semen de toros infectados naturalmente
Rossich, L.; Gutiérrez, G.; Rodriguez, Sabrina ULg et al

Poster (2008, September 22)

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