Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo

Bovine leukemia virus (BLV) is a retrovirus that naturally infects cattle. BLV integrates into the host genome as a provirus, preferentially in mature B-cells, and the infection persists through the animal lifespan. Approximately 30% of infected individuals develop a polyclonal B lymphocyte expansio...

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Autor principal:	Petersen Cruceño, Marcos Iván
Otros Autores:	Carignano, Hugo
Formato:	Tesis doctoral acceptedVersion
Lenguaje:	Español
Publicado:	Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica 2023
Materias:	Virus de leucosis bovina Carga proviral Expresión diferencial RT-qPCR RNA-seq Ciencias de la vida
Acceso en línea:	http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7140 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7140.dir/7140.PDF
Aporte de:	Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires

id	I28-R145-HWA_7140
record_format	dspace
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-145
collection	Repositorio Digital de la Universidad de Buenos Aires (UBA)
language	Español
orig_language_str_mv	spa
topic	Virus de leucosis bovina Carga proviral Expresión diferencial RT-qPCR RNA-seq Ciencias de la vida
spellingShingle	Virus de leucosis bovina Carga proviral Expresión diferencial RT-qPCR RNA-seq Ciencias de la vida Petersen Cruceño, Marcos Iván Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo
topic_facet	Virus de leucosis bovina Carga proviral Expresión diferencial RT-qPCR RNA-seq Ciencias de la vida
description	Bovine leukemia virus (BLV) is a retrovirus that naturally infects cattle. BLV integrates into the host genome as a provirus, preferentially in mature B-cells, and the infection persists through the animal lifespan. Approximately 30% of infected individuals develop a polyclonal B lymphocyte expansion, a subclinical condition called persistent lymphocytosis (PL). Among 1% to 5% of infected cattle will develop lethal B-cell lymphomas in lymph nodes or lymphosarcomas in different organs. The negative economic impact of BLV infection in dairy herds goes beyond that generated by the development of lethal lymphomas. Recent studies have shown a decrease in milk production and the productive life of infected animals, mainly those with a high proviral load (PVL).\nIn order to elucidate the underlying infection mechanism and to deepen the results obtained at the genomic level by our laboratory, in this work, we performed transcriptome studies of peripheral blood mononuclear cells (PBMCs) from naturally infected Argentine Holstein cows, which presented two contrasting proviral loads (PVL) phenotypes. This parameter has a direct relationship with disease progression and infection-spreading risk.\nIn a preliminary screening, more than 100 cows were tested by ELISA based on the positive correlation between anti-BLV antibody (Ab) levels and PVL. In a reduced number of animals, PVL was quantified by real-time PCR.\nFor this purpose, in chapter 1, a qPCR that amplifies a region of the BLV Pol gene was validated. The technique (qPCR-BLV) reliability was measured using an international reference panel. The limit of detection was established and diagnostic performance was evaluated in field samples. The qPCR-BLV was efficient in quantifying utilizing a standard curve. Thus, animals were classified phenotypically by their PVL. Then, BLV-infected animals were phenotyped as high and low PVL (HPVL and LPVL, respectively).\nIn a previous genome-wide association study (GWAS), single nucleotide polymorphisms (SNPs) were significantly associated with the PVL phenotype. In chapter 2, 8 candidate genes linked to SNPs hits were functionally characterized by assessing their expression by RT-qPCR. Analyses were carried out on 15 adult BLV-infected cows, 7 being ACPV and 8 BCPV. The study indicated differential expression (ABT1, p-value = 0.029) and significant correlations of expression levels with lymphocyte count (PRRC2A, p-value = 0.02 and IER3, p-value = 0.01), for genes not previously reported in the context of BLV infection.\nSubsequently, in chapter 3, a global transcriptome experiment using high-throughput sequencing (RNA-seq) was carried out. For this, 8 animals were selected from the 15 previously characterized, 4 per phenotype under study. After quality control, the sequencing reads obtained were mapped to the reference bovine genome, and a gene count matrix was generated for each individual. Low/no-count genes were excluded, leaving 13382 genes for the differential expression assay in PBMCs. As result, 1908 differentially expressed (DE) genes (FDR < 0.05) with a fold change (FC) ? -1.5 and ? +1.5 were obtained, of which, 774 were downregulated (DReg) in the HPVL phenotype and 1134 upregulated (UReg).\nFunctional annotation of the DE genes was performed using Gene Ontology (GO) and metabolic pathway (KEGG) terms. A functional term enrichment test was performed considering the list of expressed genes (n = 13382) ranked according to their log FC values (logFC). In addition, a protein-protein interaction (PPI) network was constructed with the DE genes (n = 1908); highly interconnected protein clusters were studied in order to identify protein complexes and metabolic pathways underlying the phenotypes studied.\nFrom the changes in the global gene expression patterns between HPVL and LPVL animals, functional terms ("Histone acetylation" (GO:0016573), "T cell-mediated immunity" (GO:0002456), "Antigen processing and presentation" (GO:0019882)) and metabolic pathways (i.e. "Th1 and Th2 cell differentiation" (bta04658), "NK cell-mediated cytotoxicity" (bta04650)) potentially related to viral gene silencing and immune response inhibition could be identified. Furthermore, a change in the expression of genes related to the mitosis process (i. e., "DNA biosynthesis" (GO:0071897), "Mitotic spindle assembly" (GO:0090307), "Kinetochore organization" (GO:0051383)) was observed in HPVL animals, that would modulate the replication of infected cell clones by promoting provirus proliferation.\nIn this work, the difference between HPVL and LPVL phenotypes was studied at the transcriptome level, in vivo in productive conditions. Due to their epidemiological and sanitary importance, the knowledge of the metabolic pathways underlying the development of HPVL in BLV-infected animals not only contributes to the identification of potential therapeutic targets but also to the identification of infection markers useful in disease control programs in herds.
author2	Carignano, Hugo
author_facet	Carignano, Hugo Petersen Cruceño, Marcos Iván
format	Tesis doctoral Tesis doctoral acceptedVersion
author	Petersen Cruceño, Marcos Iván
author_sort	Petersen Cruceño, Marcos Iván
title	Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo
title_short	Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo
title_full	Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo
title_fullStr	Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo
title_full_unstemmed	Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo
title_sort	análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por vlb en vacas de tambo
publisher	Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
publishDate	2023
url	http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7140 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7140.dir/7140.PDF
work_keys_str_mv	AT petersencrucenomarcosivan analisisdeldiferencialdetranscripcionglobalenleucocitosenrespuestaalainfeccionnaturalporvlbenvacasdetambo
_version_	1840330187893374976
spelling	I28-R145-HWA_71402025-06-26 Bovine leukemia virus (BLV) is a retrovirus that naturally infects cattle. BLV integrates into the host genome as a provirus, preferentially in mature B-cells, and the infection persists through the animal lifespan. Approximately 30% of infected individuals develop a polyclonal B lymphocyte expansion, a subclinical condition called persistent lymphocytosis (PL). Among 1% to 5% of infected cattle will develop lethal B-cell lymphomas in lymph nodes or lymphosarcomas in different organs. The negative economic impact of BLV infection in dairy herds goes beyond that generated by the development of lethal lymphomas. Recent studies have shown a decrease in milk production and the productive life of infected animals, mainly those with a high proviral load (PVL).\nIn order to elucidate the underlying infection mechanism and to deepen the results obtained at the genomic level by our laboratory, in this work, we performed transcriptome studies of peripheral blood mononuclear cells (PBMCs) from naturally infected Argentine Holstein cows, which presented two contrasting proviral loads (PVL) phenotypes. This parameter has a direct relationship with disease progression and infection-spreading risk.\nIn a preliminary screening, more than 100 cows were tested by ELISA based on the positive correlation between anti-BLV antibody (Ab) levels and PVL. In a reduced number of animals, PVL was quantified by real-time PCR.\nFor this purpose, in chapter 1, a qPCR that amplifies a region of the BLV Pol gene was validated. The technique (qPCR-BLV) reliability was measured using an international reference panel. The limit of detection was established and diagnostic performance was evaluated in field samples. The qPCR-BLV was efficient in quantifying utilizing a standard curve. Thus, animals were classified phenotypically by their PVL. Then, BLV-infected animals were phenotyped as high and low PVL (HPVL and LPVL, respectively).\nIn a previous genome-wide association study (GWAS), single nucleotide polymorphisms (SNPs) were significantly associated with the PVL phenotype. In chapter 2, 8 candidate genes linked to SNPs hits were functionally characterized by assessing their expression by RT-qPCR. Analyses were carried out on 15 adult BLV-infected cows, 7 being ACPV and 8 BCPV. The study indicated differential expression (ABT1, p-value = 0.029) and significant correlations of expression levels with lymphocyte count (PRRC2A, p-value = 0.02 and IER3, p-value = 0.01), for genes not previously reported in the context of BLV infection.\nSubsequently, in chapter 3, a global transcriptome experiment using high-throughput sequencing (RNA-seq) was carried out. For this, 8 animals were selected from the 15 previously characterized, 4 per phenotype under study. After quality control, the sequencing reads obtained were mapped to the reference bovine genome, and a gene count matrix was generated for each individual. Low/no-count genes were excluded, leaving 13382 genes for the differential expression assay in PBMCs. As result, 1908 differentially expressed (DE) genes (FDR < 0.05) with a fold change (FC) ? -1.5 and ? +1.5 were obtained, of which, 774 were downregulated (DReg) in the HPVL phenotype and 1134 upregulated (UReg).\nFunctional annotation of the DE genes was performed using Gene Ontology (GO) and metabolic pathway (KEGG) terms. A functional term enrichment test was performed considering the list of expressed genes (n = 13382) ranked according to their log FC values (logFC). In addition, a protein-protein interaction (PPI) network was constructed with the DE genes (n = 1908); highly interconnected protein clusters were studied in order to identify protein complexes and metabolic pathways underlying the phenotypes studied.\nFrom the changes in the global gene expression patterns between HPVL and LPVL animals, functional terms ("Histone acetylation" (GO:0016573), "T cell-mediated immunity" (GO:0002456), "Antigen processing and presentation" (GO:0019882)) and metabolic pathways (i.e. "Th1 and Th2 cell differentiation" (bta04658), "NK cell-mediated cytotoxicity" (bta04650)) potentially related to viral gene silencing and immune response inhibition could be identified. Furthermore, a change in the expression of genes related to the mitosis process (i. e., "DNA biosynthesis" (GO:0071897), "Mitotic spindle assembly" (GO:0090307), "Kinetochore organization" (GO:0051383)) was observed in HPVL animals, that would modulate the replication of infected cell clones by promoting provirus proliferation.\nIn this work, the difference between HPVL and LPVL phenotypes was studied at the transcriptome level, in vivo in productive conditions. Due to their epidemiological and sanitary importance, the knowledge of the metabolic pathways underlying the development of HPVL in BLV-infected animals not only contributes to the identification of potential therapeutic targets but also to the identification of infection markers useful in disease control programs in herds. Fil: Petersen Cruceño, Marcos Iván. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Carignano, Hugo Rivolta, Carina Trono, Karina Petersen Cruceño, Marcos Iván 2023-02-08 El Virus de leucosis bovina (VLB) es un retrovirus que infecta naturalmente al ganado bovino. El VLB, se integra al genoma del huésped en forma de provirus, preferentemente en las células B maduras, y la infección persiste durante toda la vida del animal. En aproximadamente el 30% de los individuos infectados se desarrolla una expansión policlonal de linfocitos B, esta condición subclínica es denominada linfocitosis persistente (LP). Entre el 1% al 5% de los bovinos infectados, desarrollan linfomas de células B en linfonódulos o linfosarcomas en distintos órganos, cuadro clínico que finaliza invariablemente con la muerte del animal. El impacto económico negativo de la infección por VLB en los rodeos lecheros trasciende al generado por el desarrollo de linfomas letales, estudios recientes han demostrado una disminución de la producción lechera y la vida productiva de los animales infectados, principalmente aquellos que presentan una alta carga proviral (CPV).\nCon el objetivo de dilucidar los mecanismos subyacentes a la infección, y profundizar resultados obtenidos a nivel genómico por nuestro laboratorio, en este trabajo se realizaron estudios a nivel de transcriptoma de células mononucleares de sangre periférica (PBMCs), de vacas Holando argentino infectadas naturalmente, que presentaban 2 fenotipos contrastantes de CPV. Este parámetro tiene una relación directa con el avance de la infección y el riesgo de contagio/diseminación de VLB en los rodeos.\nEn una fase preliminar de selección de animales (tamizaje), se analizaron más de 100 vacas por ELISA, basados en la correlación positiva entre los niveles de anticuerpos (Ac) anti VLB y la CPV. Luego, en un número menor de animales se procedió a cuantificar la CPV por PCR en tiempo real.\nPara este fin, en el capítulo 1, se validó una qPCR que amplifica una región del gen Pol del VLB. La fiabilidad de esta técnica (qPCR-VLB), se midió utilizando un panel de referencia internacional. Se estableció el límite de detección y el rendimiento diagnóstico fue evaluado en muestras de campo. La qPCR-VLB resultó eficiente para cuantificar mediante una curva estándar y, por tanto, para clasificar fenotípicamente animales por su CPV. Luego, los animales infectados con VLB fueron fenotipados como de alta y baja CPV (ACPV y BCPV, respectivamente).\nEn un estudio previo de asociación de genoma completo (GWAS), se encontraron polimorfismos de nucleótido simple (SNPs) significativamente relacionados con estos fenotipos contrastantes de CPV. En el capítulo 2, se caracterizaron funcionalmente algunos de los genes ligados a estos SNPs, evaluándose la expresión por RT-qPCR de 8 genes candidatos. Los análisis se llevaron a cabo sobre 15 vacas adultas infectadas por VLB y fenotipadas; siendo 7 ACPV y 8 BCPV. El estudio indicó expresión diferencial (ABT1, p-valor = 0,029) y correlaciones significativas de los niveles expresión con el recuento de linfocitos (PRRC2A, p-valor = 0,02 e IER3, p-valor = 0,01), para genes que no estaban previamente reportados en el contexto de infección por VLB.\nPosteriormente, en el capítulo 3, se realizó un estudio de expresión a nivel de transcriptoma global mediante secuenciación de alto rendimiento (RNA-seq). Para ello, se seleccionaron n = 8 animales de los 15 previamente caracterizados, 4 por fenotipo en estudio. Luego del control de calidad, las lecturas de secuencia obtenidas fueron mapeadas al genoma bovino de referencia y se generó la matriz de conteos de genes para cada individuo. Los genes de bajo/nulo recuento fueron excluidos, quedando 13382 genes para el ensayo de expresión diferencial en PBMCs. Como resultado, se obtuvieron 1908 genes diferencialmente expresados (FDR < 0,05) (DE) con una tasa de cambio (FC) ? -1,5 y ? +1,5, de los cuales 774 sub-expresados (DReg) en el fenotipo ACPV y 1134 sobre-expresados (UReg).\nLa anotación funcional de la lista de genes significativos, se realizó utilizando términos de ontología génica (GO), rutas metabólicas (KEGG). Un test de enriquecimiento de términos funcionales fue realizado sobre la lista de genes expresados (n = 13382), rankeados según sus valores logarítmicos de FC (logFC). Además, se llevó a cabo la construcción de una red de interacción proteína - proteína (PPI) con los genes DE significativos (n = 1908), y se buscaron grupos de proteínas altamente interconectadas (clusters) con el objetivo de identificar complejos proteicos y vías metabólicas subyacentes al fenotipo estudiado.\nA partir de los cambios en los patrones de expresión génica global entre los animales de ACPV y BCPV se pudieron identificar términos funcionales (i. e., ?Acetilación de histona? (GO:0016573), ?Inmunidad mediada por células T? (GO:0002456), ?Procesamiento y presentación de antígeno? (GO:0019882)) y rutas metabólicas (i. e., ?Diferenciación de célula Th1 y Th2? (bta04658), ?Citotoxicidad mediada por células NK? (bta04650)) potencialmente relacionados con el silenciamiento génico viral y con la inhibición de la respuesta inmune. Además, se observó un cambio en la expresión de genes relacionados con el proceso de mitosis (i. e., ?Biosíntesis del ADN? (GO:0071897), ?Ensamble del huso mitótico? (GO:0090307), "Organización del cinetocoro" (GO:0051383)) en los animales de ACPV que modularía la replicación de los clones celulares infectados promoviendo la proliferación del provirus.\nEn este trabajo, se estudió in vivo y en condiciones productivas, la diferencia entre los fenotipos de ACPV y BCPV a nivel de transcriptoma. Por su importancia a nivel epidemiológico y sanitario, el conocimiento de las vías metabólicas subyacentes al desarrollo de una ACPV en los animales infectados por VLB, contribuyen no solo a la identificación de potenciales blancos terapéuticos, sino que también pueden ser utilizadas en la identificación se marcadores de infección y en programas de control de la enfermedad en los rodeos. application/pdf Campos, Rodolfo Bratanich, Ana Cristina Panei, Javier Virus de leucosis bovina Carga proviral Expresión diferencial RT-qPCR RNA-seq spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Doctor de la Universidad de Buenos Aires en Ciencias Biológicas Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7140 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7140.dir/7140.PDF

Análisis del diferencial de transcripción global en leucocitos en respuesta a la infección natural por VLB en vacas de tambo

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