HIV quasispecies studies using ultra-deep sequencing

Abstract

The low genetic diversity of HIV in patients diagnosed with recent infection is a consequence of genetic bottleneck during transmission. However, some studies demonstrated by Sanger sequencing that this event happens less frequently in female patients, suggesting that women have greater susceptibility to be primarily infected by multiple HIV variants. Hypothetically, HIV-infected women may have a greater risk to have been infected by drug-resistant variants. Transmitted drug-resistance has major impacts in the success of the antiretroviral therapy and, thus, in AIDS progression. Nevertheless, Sanger sequencing is not able to detect resistance variants present in less than 20% of the viral population. The present study aims to evaluate the hypothesis of women lacking genetic bottleneck during HIV-1 transmission using ultra-deep sequencing. Moreover, to estimate transmitted drug-resistance prevalence in patients newly-infected by HIV-1, also using ultra-deep sequencing. Twenty female and twenty male patients recently infected by HIV-1, not under antiretroviral therapy, will be evaluated. HIV-infected men and women will be paired to age, T CD4+ cells count and HIV-1 viral load. HIV-1 viral RNA and proviral DNA will be extracted from patients´ blood samples, the viral RNA reverse transcribed, and both viral cDNA and proviral DNA submitted to nested-PCR amplification of env and pol coding-regions. Next, the PCR products will be ultra-deep sequenced, followed by phylogenetic analysis. (AU)