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Use of HIV-1 reverse transcriptase recovered from human plasma for phenotypic drug susceptibility testing
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2003 (English)In: AIDS, Vol. 17, 1463-1471 p.Article in journal (Refereed) Published
Abstract [en]

Objective: To demonstrate the use of HIV-1 reverse transcriplase (RT) recovered directly fromplasma for phenotypic drug susceptibility testing. Methods: Plasma from HIV-1 infected individuals with and without drug resistance-associated mutations were selected for the study. The blind coded plasmas were treated to inactivate cellular enzymes. The virions were immobilized on a gel and washed to remove antiretroviral drugs and RT activity blocking antibodies. The immobilized virions were lysed; the viral RTeluted and quantified, all according to the ExaVir Load procedure. The drug sensitivity profiles of each RT were determined using serially diluted drugs and modified Cavidi HS Lenti RT kits. Results: The phenotypic drug sensitivity profiles of the RT and the patterns of drug resistance mutations were highly concordant. Plasma RT from virions devoid of mutations associated with drug resistance had average 50% inhibitory concentrations (IC50) of 1.5 +/- 0.93 muM for nevirapine, 0.21 +/- 0.099 muM forefavirenz, 7.1 +/- 3.2 muM for delavirdine, 0.42 +/- 0.15 muM for azidothymidine triphosphate and 0.059 +/- 0.018 muM for didehydrothymidine triphosphate. The increase in IC50 value for RT with drugresistance associated substitutions was from 3- to more than 65-fold for non-nucleoside inhibitors and between 2- and 30-fold for thymidine analogue drugs. Conclusion: RT derived from virions recovered from the plasma of HIV infected individuals can be used foranalysis of phenotypic drug susceptibility. The methods presented provide rapid alternatives for analysingphenotypic drug susceptibility especially when the therapy is based on non-nucleoside RT inhibitors and thymidine-analogue drugs. 

Place, publisher, year, edition, pages
2003. Vol. 17, 1463-1471 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-92504DOI: 10.1097/01.aids.0000072670.21517.0dISI: 000184420900007OAI: oai:DiVA.org:uu-92504DiVA: diva2:165611
Available from: 2005-01-12 Created: 2005-01-12 Last updated: 2013-10-25Bibliographically approved
In thesis
1. Reverse Transcriptase Activity Assays for Retrovirus Quantitation and Characterization
Open this publication in new window or tab >>Reverse Transcriptase Activity Assays for Retrovirus Quantitation and Characterization
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV.

A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did.

Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data.

Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 84 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1
Medicine, retrovirus, reverse transcriptase, enzyme activity assay, MuLV, HIV, RT purification, viral load, drug susceptibility, Medicin
National Category
Dermatology and Venereal Diseases
urn:nbn:se:uu:diva-4737 (URN)91-554-6127-1 (ISBN)
Public defence
2005-02-04, Sal IV, Universitetshuset, Uppsala, 13:15
Available from: 2005-01-12 Created: 2005-01-12Bibliographically approved

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Lennerstrand, Johan
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