title: A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy.
abstract: Despite numerous studies on human immunodeficiency virus type 1 (HIV-1) fitness, many key conceptual and technical questions are still unsolved. For example, the proper system to determine virus fitness of HIV-1 is still unknown. In this study, an assay was developed to estimate HIV-1 fitness based on growth competition experiments and TaqMan real-time PCR. This novel technique was compared with several methods (i.e. virus growth kinetics, growth competition/heteroduplex-tracking analysis and single-cycle replication capacity assay) in order to analyse the impact of various genomic regions and overall genetic background on virus fitness. HIV-1 primary isolates and three different sets of recombinant viruses [i.e. recombinant clones carrying protease (PR), reverse transcriptase (RT) or the 3' end of Gag, PR and RT (3'Gag/PR/RT), sequences amplified by PCR from the same primary isolates)] were evaluated. Here, it is demonstrated that, in spite of intrinsic differences, both growth competition/TaqMan and single-cycle replication assays detected a significant reduction in HIV-1 fitness as a consequence of drug-resistant mutations in pol. However, this new assay, based on HIV-1 isolates, may be useful to quantify replicative fitness in viruses from patients treated simultaneously with antiretroviral drugs targeting different genomic regions of HIV-1 (e.g. pol and env).
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Weber
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Hector R
authors.initials: HR
authors.lastname: Rangel
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Bikram
authors.initials: B
authors.lastname: Chakraborty
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Mahlet
authors.initials: M
authors.lastname: Tadele
authors.affiliation: Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari Germans Trias I Pujol, Badalona, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Martinez
authors.affiliation: Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari Germans Trias I Pujol, Badalona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Martinez-Picado
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Marotta
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Muneer
authors.initials: M
authors.lastname: Mirza
authors.affiliation: Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari Germans Trias I Pujol, Badalona, Spain.
authors.forename: Lidia
authors.initials: L
authors.lastname: Ruiz
authors.affiliation: Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari Germans Trias I Pujol, Badalona, Spain.
authors.forename: Bonaventura
authors.initials: B
authors.lastname: Clotet
authors.affiliation: ViroLogic Inc., South San Francisco, CA, USA.
authors.forename: Terri
authors.initials: T
authors.lastname: Wrin
authors.affiliation: ViroLogic Inc., South San Francisco, CA, USA.
authors.forename: Christos J
authors.initials: CJ
authors.lastname: Petropoulos
authors.affiliation: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, NN10, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: Miguel E
authors.initials: ME
authors.lastname: Quiñones-Mateu
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics; D004359:Drug Therapy, Combination; D015686:Gene Products, env / Q000235:genetics; D015684:Gene Products, pol / Q000235:genetics; D015658:HIV Infections / Q000150:complications / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000187:drug effects / Q000254:growth & development* / Q000502:physiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D019914:Taq Polymerase / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D019380:Anti-HIV Agents; D015686:Gene Products, env; D015684:Gene Products, pol; D018894:Reverse Transcriptase Inhibitors; D019914:Taq Polymerase