title: Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.
abstract: Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV delta BstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates.
authors.affiliation: Antiviral Therapeutic Research Unit, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.
authors.forename: P
authors.initials: P
authors.lastname: Kellam
authors.forename: B A
authors.initials: BA
authors.lastname: Larder
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D015704:CD4 Antigens / Q000032:analysis; D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis; D005838:Genotype; D015658:HIV Infections / Q000382:microbiology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism / Q000382:microbiology; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D010948:Viral Plaque Assay; D014779:Virus Replication / Q000187:drug effects; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D015704:CD4 Antigens; D004279:DNA, Viral; D015215:Zidovudine