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

Background: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. Principal Findings: Here, 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 minorit

A Stochastic Model of Latently Infected Cell Reactivation and Viral Blip Generation in Treated HIV Patients

Motivated by viral persistence in HIV+ patients on long-term anti-retroviral treatment (ART), we present a stochastic model of HIV viral dynamics in the blood stream. We consider the hypothesis that the residual viremia in patients on ART can be explained principally by the activation of cells latently infected by HIV before the initiation of ART and that viral blips (clinically-observed short periods of detectable viral load) represent large deviations from the mean. We model the system as a continuous-time, multi-type branching process. Deriving equations for the probability generating function we use a novel numerical approach to extract the probability distributions for latent reservoir sizes and viral loads. We find that latent reservoir extinction-time distributions underscore the importance of considering reservoir dynamics beyond simply the half-life. We calculate blip amplitudes and frequencies by computing complete viral load probability distributions, and study the duration of viral blips via direct numerical simulation. We find that our model qualitatively reproduces short small-amplitude blips detected in clinical studies of treated HIV infection. Stochastic models of this type provide insight into treatment-outcome variability that cannot be found from deterministic models

Reduced evolutionary rates in HIV-1 reveal extensive latency periods among replicating lineages

BACKGROUND: HIV-1 can persist for the duration of a patient’s life due in part to its ability to hide from the immune system, and from antiretroviral drugs, in long-lived latent reservoirs. Latent forms of HIV-1 may also be disproportionally involved in transmission. Thus, it is important to detect and quantify latency in the HIV-1 life cycle. RESULTS: We developed a novel molecular clock–based phylogenetic tool to investigate the prevalence of HIV-1 lineages that have experienced latency. The method removes alternative sources that may affect evolutionary rates, such as hypermutation, recombination, and selection, to reveal the contribution of generation-time effects caused by latency. Our method was able to recover latent lineages with high specificity and sensitivity, and low false discovery rates, even on relatively short branches on simulated phylogenies. Applying the tool to HIV-1 sequences from 26 patients, we show that the majority of phylogenetic lineages have been affected by generation-time effects in every patient type, whether untreated, elite controller, or under effective or failing treatment. Furthermore, we discovered extensive effects of latency in sequence data (gag, pol, and env) from reservoirs as well as in the replicating plasma population. To better understand our phylogenetic findings, we developed a dynamic model of virus-host interactions to investigate the proportion of lineages in the actively replicating population that have ever been latent. Assuming neutral evolution, our dynamic modeling showed that under most parameter conditions, it is possible for a few activated latent viruses to propagate so that in time, most HIV-1 lineages will have been latent at some time in their past. CONCLUSIONS: These results suggest that cycling in and out of latency plays a major role in the evolution of HIV-1. Thus, no aspect of HIV-1 evolution can be fully understood without considering latency - including treatment, drug resistance, immune evasion, transmission, and pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-014-0081-0) contains supplementary material, which is available to authorized users