A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance

A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1–280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope ( env ) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using Apa I or Age I and Aar I restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination ( R 2 ) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 × 10 4 cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

A single-loop recombinant pseudotyped-virus-based assay to detect HIV-1 phenotypic resistance

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Publisher
Springer Vienna
Copyright
Copyright © 2015 by Springer-Verlag Wien
Subject
Biomedicine; Virology; Medical Microbiology; Infectious Diseases
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-015-2386-2
Publisher site
See Article on Publisher Site

Abstract

HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1–280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope ( env ) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using Apa I or Age I and Aar I restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination ( R 2 ) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 × 10 4 cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents.

Journal

Archives of VirologySpringer Journals

Published: Jun 1, 2015

References

  • HIV drug resistance
    Clavel, F; Hance, AJ
  • Genotypic and phenotypic resistance testing of HIV-1 in routine clinical care
    Hirsch, HH; Drechsler, H; Holbro, A; Hamy, F; Sendi, P; Petrovic, K; Klimkait, T; Battegay, M
  • Inclusion of full length human immunodeficiency virus type 1 (HIV-1) gag sequences in viral recombinants applied to drug susceptibility phenotyping
    Robinson, LH; Gale, CV; Kleim, JP
  • Resistance to HIV protease inhibitors
    Condra, JH
  • Genotypic and phenotypic resistance testing of HIV-1 in routine clinical care
    Hirsch, HH; Drechsler, H; Holbro, A; Hamy, F; Sendi, P; Petrovic, K; Klimkait, T; Battegay, M

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