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Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogeneous group of HIV type 1 subtype B viruses with a heterogeneous group of divergent strains
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
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1998 In: AIDS Res Hum Retroviruses, Vol. 14, no 4, 347-352 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
1998. Vol. 14, no 4, 347-352 p.
URN: urn:nbn:se:uu:diva-92501OAI: oai:DiVA.org:uu-92501DiVA: diva2:165608
Available from: 2005-01-12 Created: 2005-01-12Bibliographically 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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