Estudo das quasispecies do HIV-1 com o uso de sequenciamento paralelo maciço
Arquivos
Data
2015-07-13
Autores
Leda, Ana Rachel Oliveira 
Orientadores
Diaz, Ricardo Sobhie
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
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Resumo
A transmissão do HIV-1 é caracterizada por uma intensa restrição genética, na qual a infecção é normalmente estabelecida por uma única variante viral, capaz de evoluir rapidamente e moldar a progressão para a doença. O objetivo deste estudo é caracte-rizar profundamente a evolução do HIV-1 durante o primeiro ano de infecção em paci-entes recém-infectados e correlacionar a evolução viral com progressão da doença, tropismo e infecção por variantes virais resistentes aos antirretrovirais. Amostras de RNA e DNA de 25 pacientes foram amplificadas independentemente para as regiões pol e env do HIV-1 e submetidas ao sequenciamento paralelo maciço. A infecção por um única variante foi observada em 92% da população do estudo. A diversidade ge-nética e a divergência diferiram entre as regiões genômicas do vírus avaliadas, sendo que a região V3 do envelope apresentou as maiores taxas. A diversidade genética encontrada no PBMC era maior em comparação ao plamas, sugerindo compartimen-talização do vírus. Os níveis de pressão seletiva positiva eram maiores na região V3, em relação as demais regiões. Baixos níveis de diversidade genética foram vistos acompanhados de baixos ou elevados níveis de replicação viral, enquanto que eleva-dos níveis de diversidade genética foram vistos acompanhados de níveis intermediá-rios de replicação viral. Nossos resultados sugerem que baixos níveis de diversidade genética durante a infecção recente são uma consequência de um sistema imunitário muito ou pouco eficiente. E ainda, as pressões seletivas durante o primeiro ano de infecção estão focadas principalmente no envelope viral, sugerindo que respostas imunitárias humorais tem grande importância para a progressão da doença.
The transmission of HIV-1 is characterized by a severe genetic bottleneck, in which an infection is typically established by a single variant, that evolves rapidly and shapes disease progression. The present study aimed to deeply characterize the HIV-1 evolu-tion during the first year of infection among recently-infected patients from Brazil, and correlate this evolution with disease progression, viral tropism and infection by trans-mitted drug resistance variants, using ultra-deep sequencing technology. RNA and DNA samples from twenty-five patients were independently amplified for pol and env genes regions and deep sequenced using 454 technology. Infection an unique HIV-1 strains occurred in 92% of our study population. Genetic diversity and divergence dif-fered among genomic regions, with greater differences found at the V3 region of the envelope. The genetic diversity in the PBMC was higher than in the plasma for all ana-lyzed regions, suggesting viral compartmentalization. Positive selective pressure was greater for the V3 region, compared to pol gene regions. We observed that low levels of genetic diverdity was accompanied by either low or high levels of viral replication, depending on the efficiency of the immune responses. Low levels of CD4+ T cell was associated with poor immune responses against HIV-1, which culminates in an also poor selective pressure, represented by the low levels of genetic diversity. We propose that low levels of viral diversification during recent infection are a consequence of ei-ther a very effective or a very ineffective host immune system. Additionally, immune selective pressures during the first year of infection is mainly focused on the envelope, suggesting that humoral immune responses have an important role in mounting the host´s immune responses that will shape viral diversity and disease progression.
The transmission of HIV-1 is characterized by a severe genetic bottleneck, in which an infection is typically established by a single variant, that evolves rapidly and shapes disease progression. The present study aimed to deeply characterize the HIV-1 evolu-tion during the first year of infection among recently-infected patients from Brazil, and correlate this evolution with disease progression, viral tropism and infection by trans-mitted drug resistance variants, using ultra-deep sequencing technology. RNA and DNA samples from twenty-five patients were independently amplified for pol and env genes regions and deep sequenced using 454 technology. Infection an unique HIV-1 strains occurred in 92% of our study population. Genetic diversity and divergence dif-fered among genomic regions, with greater differences found at the V3 region of the envelope. The genetic diversity in the PBMC was higher than in the plasma for all ana-lyzed regions, suggesting viral compartmentalization. Positive selective pressure was greater for the V3 region, compared to pol gene regions. We observed that low levels of genetic diverdity was accompanied by either low or high levels of viral replication, depending on the efficiency of the immune responses. Low levels of CD4+ T cell was associated with poor immune responses against HIV-1, which culminates in an also poor selective pressure, represented by the low levels of genetic diversity. We propose that low levels of viral diversification during recent infection are a consequence of ei-ther a very effective or a very ineffective host immune system. Additionally, immune selective pressures during the first year of infection is mainly focused on the envelope, suggesting that humoral immune responses have an important role in mounting the host´s immune responses that will shape viral diversity and disease progression.
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Citação
LEDA, Ana Rachel Oliveira. Estudo das quasispecies do HIV-1 com o uso de sequenciamento paralelo maciço. 2015. 130 f. Tese (Doutorado em Infectologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.