title: Novel two-round phenotypic assay for protease inhibitor susceptibility testing of recombinant and primary HIV-1 isolates.
abstract: Antiretroviral drug susceptibility tests facilitate therapeutic management of HIV-1-infected patients. Although genotyping systems are affordable, inaccuracy in the interpretation of complex mutational patterns may limit their usefulness. Currently available HIV-1 phenotypic assays are based on the generation of recombinant viruses in which the specific viral gene of interest, derived from a patient plasma sample, is cloned into a susceptible genetic viral backbone prior to in vitro drug susceptibility evaluation. Nevertheless, in the case of protease inhibitors, not only are mutations in the HIV-1 protease-coding region involved in resistance, but the role of Gag in drug susceptibility has also recently been reported. In order to avoid the inherent limitations resulting from partial cloning of the viral genome, we designed and evaluated a new experimental strategy to test the in vitro susceptibility of primary viral isolates to protease inhibitors. Our protocol, which is based on a two-round infection protocol using the reporter TZM-bl cell line, showed a good correlation with genotypic resistance prediction and with the Antivirogram phenotypic assay, in both protease-recombinant viruses and primary viral isolates. The protocol is suitable for any HIV-1 subtype and enables rapid in-house measurement of protease inhibitor susceptibility, thus making it possible to evaluate the concomitant effects of both patient-derived gag and protease-coding regions.
authors.affiliation: AIDS Research Institute IrsiCaixa, Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Puertas
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Buzón
authors.forename: Mònica
authors.initials: M
authors.lastname: Ballestero
authors.forename: Peter
authors.initials: P
authors.lastname: Van Den Eede
authors.forename: Bonaventura
authors.initials: B
authors.lastname: Clotet
authors.forename: Julia G
authors.initials: JG
authors.lastname: Prado
authors.forename: Javier
authors.initials: J
authors.lastname: Martinez-Picado
mesh_terms: D015658:HIV Infections / Q000821:virology; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D011995:Recombination, Genetic
chemical_list: D017320:HIV Protease Inhibitors