Human immunodeficiency virus type 1 drug susceptibility determination by using recombinant viruses generated from patient sera tested in a cell-killing assay.
A simple approach for the determination of drug susceptibilities by using human immunodeficiency virus type 1 (HIV-1) RNA from the sera of patients is described. HIV-1 RNA was extracted from patient sera, and the 5' part of the reverse transcriptase (RT) gene was transcribed into DNA and amplified in a nested PCR. The amplified fragment covers the 3' part of the protease gene and amino acids 1 to 304 of the RT gene. This fragment can be introduced through homologous recombination, as described previously, into a novel HIV-1 reference strain (pHXB2 delta 2-261RT) from which amino acids 2 to 261 of RT have been deleted. The resulting recombinant virus expresses all properties of the HXB2 reference strain except for those encoded by the introduced part of the patient RT gene. Recombinant viruses were subsequently tested for drug susceptibility in a microtiter format killing assay [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay] as well as in the standard HeLa CD4+ plaque reduction assay. Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from patients receiving alternating nevirapine and zidovudine treatment or lamivudine-zidovudine combination therapy. In conclusion, this approach enables high-through-put determination of the drug susceptibilities of serum RNA-derived RT genes, independent of the patient's viral background, and generates the possibility of relating changes in susceptibility to changes in viral genotypes.
Department of Virology, Eijkman-Winkler Institute, University Hospital Utrecht, The Netherlands. c.boucher@lab.azu.nl
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Keulen
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van Bommel
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Nijhuis
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de Jong
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Schipper
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D019380:Anti-HIV Agents / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002460:Cell Line; D003001:Cloning, Molecular; D018274:Electroporation; D015658:HIV Infections / Q000097:blood / Q000821:virology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000096:biosynthesis; D011995:Recombination, Genetic; D013778:Tetrazolium Salts; D013844:Thiazoles; D010948:Viral Plaque Assay
D019380:Anti-HIV Agents; D012367:RNA, Viral; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue