Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing

Abstract

BackgroundUltra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing.Principal FindingsHere, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180–220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406–35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07–0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs.ConclusionsWith this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.

The Rate of Immune Escape Vanishes When Multiple Immune Responses Control an HIV Infection

“…Recently developed sequencing methods have made it possible to sequence the whole HIV quasispecies (7)(8)(9)(10)(11). Variants can now be detected even when their frequency in the quasispecies is as low as 0.05% (7).…”

“…Variants can now be detected even when their frequency in the quasispecies is as low as 0.05% (7). Typically, HIV infections start with a single founder virus, which drastically increases in diversity around the peak in the viral load (9,10).…”

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“…Existe un grupo de mutaciones que son frecuentes en las poblaciones minoritarias y que, por su relevancia al relacionarse con la resistencia a determinados medicamentos, probablemente constituirá un problema para estos pacientes 17,18 . Los resultados de la CV elevada y de recuentos bajos de LTCD4+ en pacientes tratados indican un fallo a la terapia de largo tiempo de evolución 19 .…”

Optimization of dengue virus genome assembling using GSFLX 454 pyrosequencing data: evaluation of assembling strategies

“…In the past, efforts have been made to use NGS-based approaches, in particular pyrosequencing, for obtaining genetic data from different viral pathogens, such as HIV; avian leukosis virus; lyssaviruses; enteric viruses, and few arboviruses, including DENV-1 and phleboviruses (Victoria et al, 2009;Bimber et al, 2010;Bishop-Lilly et al, 2010;Day et al, 2010;Hedskog et al, 2010;De Wolf et al, 2011;Schinazi et al, 2011;Abbate et al, 2011;De Benedictis et al, 2011;Palacios et al, 2011).…”