HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 DRUG SUSCEPTIBILITY DETERMINATIONBY USING RECOMBINANT VIRUSES GENERATED FROM PATIENT SERA TESTED IN A CELL-KILLING ASSAY

A simple approach for the determination of drug susceptibilities by us ing human immunodeficiency virus type 1 (HIV-1) RNA from the sera of p atients is described. HIV-1 RNA was extracted from patient sera, and t he 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 301 of the RT gene, This fragment can be introduced through homologous recombination, as d escribed 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 refer ence strain except for those encoded by the introduced part of the pat ient RT gene, Recombinant viruses were subsequently tested for drug su sceptibility in a microtiter format killing assay -(4,5-dimethylthiazo l-2-yl)-2,5-diphenltetrazolium bromide assay] as well as in the standa rd HeLa CD4(+) plaque reduction assay, Similar susceptibility profiles were obtained by each assay with recombinant viruses derived from pat ients receiving alternating nevirapine and zidovudine treatment or lam ivudine-zidovudine combination therapy, In conclusion, this approach e nables high-throughput determination of the drug susceptibilities of s erum RNA-derived RT genes, independent of the patient's viral backgrou nd, and generates the possibility of relating changes in susceptibilit y to changes in viral genotypes.