Caracterização molecular do vírus linfotrópico humano de células T, genoma completo e parcial através do sequenciamento de última geração pela plataforma Illumina
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Data
2015-01-31
Autores
Farias, Rodrigo Pessoa de [UNIFESP]
Orientadores
Al Sanabani, Sabri Saeed Mohammed Ahmed [UNIFESP]
Tipo
Dissertação de mestrado
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Introdução: Nesse trabalho relatamos a variabilidade das sequencias do Vírus Linfotrópico de Célula Humana T tipo 1 (HTLV-1) genoma completo e parcial de 90 indivíduos, destes sendo 48 portadores assintomáticos, 35 pacientes com Paraparesia Espástica tropical/Mielopatia associada ao HTLV-1 (HAM/TSP) e 7 pacientes portadores de leucemia/linfoma de células T adulto, utilizando o sequenciamento de nova geração do aparelho da Illumina (MiSeq). Metodologia: Foram coletadas amostras de sangue dos 90 indivíduos. O DNA foi extraído a partir das células mononucleares do sangue periférico PBMC para medir a carga proviral e amplificar o HTLV-1 em dois fragmentos que se sobrepõem. Os produtos de PCR amplificados foram sequenciados no sequenciador de nova geração MiSeq (Illumina). Os dados gerados foram montados, alinhados e mapeados contra um genoma de HTLV-1 já descrito com semelhança genética para posteriores análises filogenéticas. Resultados: O sequenciamento de alto rendimento por síntese foi utilizado para obter uma média de cobertura de 3210-5200 para cada amostra, obtendo-se genomas parciais (n=14) e genomas completos (n=76) do HTLV-1. Os resultados, utilizando-se as sequencias geradas pelo sequenciamento e baseado em árvores filogenéticas, revelaram que 86 (95,5%) indivíduos são infectados com o grupo A transcontinental e subtipo A cosmopolita (aA) e 4 (4,5%) indivíduos com o subtipo B japonês (aB). Uma comparação entre os ácidos nucléicos e aminoácidos dos genomas completos nas três categorias não apresentou nenhuma relação entre o genótipo e as condições clínicas dos indivíduos. As relações evolutivas entre as sequências do HTLV-1 foram descritas a partir de sequências de nucleotídeos, e estes resultados são consistentes suportando a hipótese de que houve várias entradas do subtipo transcontinental no Brasil. Conclusões: Este estudo aumenta o número de sequências de genomas completos do HTLV-1 disponíveis em banco de dados do subtipo aA de 8 para 81 e aB de 2 para 5. Nossos dados confirmam que o subtipo transcontinental cosmopolita é o mais prevalente na população brasileira. Assim espera-se que as sequencias geradas irão acrescentar a nossa compreensão atual da história evolutiva deste vírus.
Background: Here, we report on the partial and full-length genomic (FLG) variability of HTLV-1 sequences from 90 well-characterized subjects, including 48 HTLV-1 asymptomatic carriers (ACs), 35 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and 7 adult T-cell leukemia/lymphoma (ATLL) patients, using an Illumina paired-end protocol. Methods: Blood samples were collected from 90 individuals, and DNA was extracted from the PBMCs to measure the proviral load and to amplify the HTLV-1 FLG from two overlapping fragments. The amplified PCR products were subjected to deep sequencing. The sequencing data were assembled, aligned, and mapped against the HTLV-1 genome with sufficient genetic resemblance and utilized for further phylogenetic analysis. Results: A high-throughput sequencing-by-synthesis instrument was used to obtain an average of 3210- and 5200-fold coverage of the partial (n = 14) and FLG (n = 76) data from the HTLV-1 strains, respectively. The results based on the phylogenetic trees of consensus sequences from partial and FLGs revealed that 86 (95.5%) individuals were infected with the transcontinental sub-subtypes of the cosmopolitan subtype (aA) and that 4 individuals (4.5%) were infected with the Japanese sub-subtypes (aB). A comparison of the nucleotide and amino acids of the FLG between the three clinical settings yielded no correlation between the sequenced genotype and clinical outcomes. The evolutionary relationships among the HTLV sequences were inferred from nucleotide sequence, and the results are consistent with the hypothesis that there were multiple introductions of the transcontinental subtype in Brazil. Conclusions: This study has increased the number of subtype aA full-length genomes from 8 to 81 and HTLV-1 aB from 2 to 5 sequences. The overall data confirmed that the cosmopolitan transcontinental sub-subtypes were the most prevalent in the Brazilian population. It is hoped that this valuable genomic data will add to our current understanding of the evolutionary history of this medically important virus.
Background: Here, we report on the partial and full-length genomic (FLG) variability of HTLV-1 sequences from 90 well-characterized subjects, including 48 HTLV-1 asymptomatic carriers (ACs), 35 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and 7 adult T-cell leukemia/lymphoma (ATLL) patients, using an Illumina paired-end protocol. Methods: Blood samples were collected from 90 individuals, and DNA was extracted from the PBMCs to measure the proviral load and to amplify the HTLV-1 FLG from two overlapping fragments. The amplified PCR products were subjected to deep sequencing. The sequencing data were assembled, aligned, and mapped against the HTLV-1 genome with sufficient genetic resemblance and utilized for further phylogenetic analysis. Results: A high-throughput sequencing-by-synthesis instrument was used to obtain an average of 3210- and 5200-fold coverage of the partial (n = 14) and FLG (n = 76) data from the HTLV-1 strains, respectively. The results based on the phylogenetic trees of consensus sequences from partial and FLGs revealed that 86 (95.5%) individuals were infected with the transcontinental sub-subtypes of the cosmopolitan subtype (aA) and that 4 individuals (4.5%) were infected with the Japanese sub-subtypes (aB). A comparison of the nucleotide and amino acids of the FLG between the three clinical settings yielded no correlation between the sequenced genotype and clinical outcomes. The evolutionary relationships among the HTLV sequences were inferred from nucleotide sequence, and the results are consistent with the hypothesis that there were multiple introductions of the transcontinental subtype in Brazil. Conclusions: This study has increased the number of subtype aA full-length genomes from 8 to 81 and HTLV-1 aB from 2 to 5 sequences. The overall data confirmed that the cosmopolitan transcontinental sub-subtypes were the most prevalent in the Brazilian population. It is hoped that this valuable genomic data will add to our current understanding of the evolutionary history of this medically important virus.
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Citação
FARIAS, Rodrigo Pessoa de. Caracterização molecular do vírus linfotrópico humano de células T, genoma completo e parcial através do sequenciamento de última geração pela
plataforma Illumina. 2015. Dissertação (Mestrado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.