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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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2023
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| 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 |
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| Sumario: | 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. |
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