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A Rapid Method for Simultaneous Detection of Phenotypic Resistance to Inhibitors of Protease and Reverse Transcriptase in Recombinant Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Antiretroviral Drugs
Open accessAbstract
Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HIV type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR- and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4 + T lymphocytes (MT4) with the pGEMT3ΔPRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell–3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system that allows high sample throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay system facilitates the rapid large-scale phenotypic resistance determinations for all RT and PR inhibitors in one standardized assay.
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Citations
Cited by
Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database
Shafer, R. W., Stevenson, D., & Chan, B. (1999) · Nucleic Acids Research · doi:10.1093/nar/27.1.348 · Cited by 907
Hirsch, M. S., Günthard, H. F., Schapiro, J. M., Brun‐Vézinet, F., Clotet, B., Hammer, S. M., Johnson, V. A., Kuritzkes, D. R., Mellors, J. W., Pillay, D., et al. (2008) · Clinical Infectious Diseases · doi:10.1086/589297 · Cited by 573
HIV-1 Drug Resistance in Newly Infected Individuals
Boden, D. (1999) · JAMA · doi:10.1001/jama.282.12.1135 · Cited by 466
Saleem, M., & Saeed, M. T. (2020) · Journal of King Saud University - Science · doi:10.1016/j.jksus.2019.02.013 · Cited by 147
Metadata
- Authors
- Hertogs, K., de Béthune, M.-P., Miller, V., Ivens, T., Schel, P., Van Cauwenberge, A., Van den Eynde, C., van Gerwen, V., Azijn, H., van Houtte, M., Peeters, F., Staszewski, S., Conant, M., Bloor, S., Kemp, S., Larder, B., & Pauwels, R.
- Venue
- Antimicrobial Agents and Chemotherapy
- Publisher
- American Society for Microbiology
- Published
- 1998-02
- Volume
- 42
- Issue
- 2
- Pages
- 269-276
- ISSN
- 0066-4804 (ISSN-L)
- 1098-6596
- OpenAlex
- W2134713579
- Cited by
- 389
- References
- 0
- FWCI
- 28.32
- Type
- journal article
- Language
- en
- Open access
- green
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- The paper
- Hertogs, K., de Béthune, M.-P., Miller, V., Ivens, T., Schel, P., Van Cauwenberge, A., Van den Eynde, C., van Gerwen, V., Azijn, H., van Houtte, M., Peeters, F., Staszewski, S., Conant, M., Bloor, S., Kemp, S., Larder, B., & Pauwels, R. (1998). A Rapid Method for Simultaneous Detection of Phenotypic Resistance to Inhibitors of Protease and Reverse Transcriptase in Recombinant Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Antiretroviral Drugs. Antimicrobial Agents and Chemotherapy. https://doi.org/10.1128/aac.42.2.269
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
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