title: Dynamics of HIV-1 quasispecies during antiviral treatment dissected using ultra-deep pyrosequencing.
abstract: BACKGROUND
  Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing.
  
  
  RESULTS
  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 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.
  
  
  CONCLUSIONS
  With 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.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Hedskog
authors.forename: Mattias
authors.initials: M
authors.lastname: Mild
authors.forename: Johanna
authors.initials: J
authors.lastname: Jernberg
authors.forename: Ellen
authors.initials: E
authors.lastname: Sherwood
authors.forename: Göran
authors.initials: G
authors.lastname: Bratt
authors.forename: Thomas
authors.initials: T
authors.lastname: Leitner
authors.forename: Joakim
authors.initials: J
authors.lastname: Lundeberg
authors.forename: Björn
authors.initials: B
authors.lastname: Andersson
authors.forename: Jan
authors.initials: J
authors.lastname: Albert
mesh_terms: D019380:Anti-HIV Agents / Q000627:therapeutic use; D024882:Drug Resistance, Viral / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000145:classification* / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D019380:Anti-HIV Agents