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Journal of Clinical Microbiology, July 2009, p. 2232-2242, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.01739-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs{triangledown}

Kris Covens,1* Nathalie Dekeersmaeker,1 Yoeri Schrooten,1,2 Jan Weber,3,{dagger} Dominique Schols,1 Miguel E. Quiñones-Mateu,3,{dagger} Anne-Mieke Vandamme,1 and Kristel Van Laethem1,2

Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium,1 AIDS Reference Laboratory, University Hospitals Leuven, Leuven, Belgium,2 Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio3

Received 9 September 2008/ Returned for modification 14 March 2009/ Accepted 21 April 2009

Combination therapy can successfully suppress human immunodeficiency virus (HIV) replication in patients but selects for drug resistance, requiring subsequent resistance-guided therapeutic changes. This report describes the development and validation of a novel assay that offers a uniform method to measure susceptibility to all clinically approved HIV type 1 (HIV-1) drugs targeting reverse transcriptase (RT), protease (PR), integrase (IN), and viral entry. It is an assay in which the antiviral effect on infection within a single replication cycle is measured in triply transfected U87.CD4.CXCR4.CCR5 cells, based on homologous recombination between patient-derived amplicons and molecular proviral clones tagged with the enhanced green fluorescent protein (EGFP) reporter gene and from which certain viral genomic regions are removed. The deletions stretch from p17 codon 7 to PR codon 98 in pNL4.3-{Delta}gagPR-EGFP, from PR codons 1 to 99 in pNL4.3-{Delta}PR-EGFP, from RT codons 1 to 560 in pNL4.3-{Delta}RT-EGFP, from IN codons 1 to 288 in pNL4.3-{Delta}IN-EGFP, and from gp120 codon 34 to gp41 codon 237 in pNL4.3-{Delta}env-EGFP. The optimized experimental conditions enable the investigation of patient samples regardless of viral subtype or coreceptor use. The extraction and amplification success rate for a set of clinical samples belonging to a broad range of HIV-1 group M genetic forms (A-J, CRF01-03, CRF05, and CRF12-13) and displaying a viral load range of 200 to >500,000 RNA copies/ml was 97%. The drug susceptibility measurements, based on discrimination between infected and noninfected cells on a single-cell level by flow cytometry, were reproducible, with coefficients of variation for resistance ranging from 7% to 31%, and were consistent with scientific literature in terms of magnitude and specificity.

* Corresponding author. Mailing address: Rega Institute for Medical Research, Microbiology and Immunology, Clinical and Epidemiological Virology, Minderbroedersstraat 10, 3000 Leuven, Belgium. Phone: 32-16-332177. Fax: 32-16-332131. E-mail: kris.covens{at}uz.kuleuven.ac.be

{triangledown} Published ahead of print on 29 April 2009.

{dagger} Present address: Diagnostic Hybrids, Inc., Cleveland, OH.

Journal of Clinical Microbiology, July 2009, p. 2232-2242, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.01739-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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