Advertisement

Molecular Diagnosis & Therapy

, Volume 15, Issue 5, pp 293–301 | Cite as

Phenotypic Analysis of HIV-1 Genotypic Drug-Resistant Isolates from China, Using a Single-Cycle System

  • Zheng Jia
  • Sihong Xu
  • Jianhui Nie
  • Jingyun Li
  • Ping Zhong
  • Wenbo Wang
  • Youchun Wang
Original Research Article

Abstract

Background and Objectives: Drug resistance in HIV-1 is one of the main causes of failure of antiretroviral therapy. Phenotypic detection of drug-resistant HIV-1 can provide guidance in selecting the optimal treatment regimen. Traditional phenotype assays are labor intensive and time consuming. Thus, a rapid and convenient phenotype assay with a single cycle of replication was developed and used in this study.

Methods: Two restriction endonuclease sites, ApaI and AgeI, were inserted into the plasmid pSG3,DΔenv using site-directed mutagenesis. The reverse transcriptase and protease genes of HIV-1 were amplified from patients and cloned into the modified pSG3Δenv. Sixteen original recombinant pseudoviruses were generated. The phenotypic susceptibility of these 16 recombinant pseudoviruses to 12 antiretro viral drugs was determined using a luciferase reporter system, and the phenotype and genotype results were compared.

Results: A modified phenotype assay with a single-cycle system was established, and its reproducibility and feasibility were validated. Approximately 89% of the phenotype results were in agreement with the genotype results; this slight disagreement may have been due to complex and multiple resistance mutations. The phenotype results showed that individual pseudoviruses with four thymidine analog mutations (TAMs) [M41L, T67N, L210W, and T215Y] in combination with various other mutations had different levels of resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Mutations E44A, T69D, and V118I influenced the pattern of resistance of TAMs. The level of resistance to non-NRTIs (NNRTIs) was also variable when different NNRTI-resistance mutations were combined.

Conclusion: The single-cycle pseudovirus phenotypic susceptibility detection system reflects HIV-1 drug resistance, especially for complex resistance mutants, and could be used to screen new antiretroviral candidates.

Keywords

Polymerase Chain Reaction Product ApaI Restriction Endonuclease Site Genotype Result Phenotype Result 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

The authors would like to thank Mr Zuoyi Bao and Dr Jue Li from the Institute of Microbiology and Epidemiology at the Academy of Military Medical Science (Beijing, P.R. China) and Mr Yile Xue from the Shanghai Center of Disease Control (Shanghai, P.R. China) for their technical assistance, and Dr Fujie Zhang from the National Center of Disease Control (Beijing, P.R. China) for his suggestions.

This study was supported by the Key Project on Infectious Diseases such as AIDS, Hepatitis, and Tuberculosis (grant no. 2009ZX10004-801) from the Ministry of Science and Technology (Beijing, P.R. China). The authors declare no conflicts of interest that are directly relevant to the content of this manuscript.

References

  1. 1.
    Mohammadpour A, Yekta ZP, Nikbakht Nasrabadi AR. HIV-infected patients' adherence to highly active antiretroviral therapy: a phenomeno-logical study. Nurs Health Sci 2010; 12: 464–9PubMedCrossRefGoogle Scholar
  2. 2.
    Grover D, Copas A, Green H, et al. What is the risk of mortality following diagnosis of multidrug-resistant HIV-1? J Antimicrob Chemother 2008; 61: 705–13PubMedCrossRefGoogle Scholar
  3. 3.
    Gallant JE. Antiretroviral drug resistance and resistance testing. Top HIV Med 2005; 13: 138–42PubMedGoogle Scholar
  4. 4.
    Hertogs K, de Bethune MP, Miller V, et al. 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. Antimicrob Agents Chemother 1998; 42: 269–76PubMedCrossRefGoogle Scholar
  5. 5.
    Petropoulos CJ, Parkin NT, Limoli KL, et al. A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1. Antimicrob Agents Chemother 2000; 44: 920–8PubMedCrossRefGoogle Scholar
  6. 6.
    Zhang H, Zhou Y, Alcock C, et al. Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1. J Virol 2004; 78: 1718–29PubMedCrossRefGoogle Scholar
  7. 7.
    McMahon MA, Shen L, Siliciano RF. New approaches for quantitating the inhibition of HIV-1 replication by antiviral drugs in vitro and in vivo. Curr Opin Infect Dis 2009; 22: 574–82PubMedCrossRefGoogle Scholar
  8. 8.
    Covens K, Dekeersmaeker N, Schrooten Y, et al. Novel recombinant virus assay for measuring susceptibility of human immunodeficiency virus type 1 group M subtypes to clinically approved drugs. J Clin Microbiol 2009; 47: 2232–42PubMedCrossRefGoogle Scholar
  9. 9.
    Kutner RH, Zhang XY, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 2009; 4: 495–505PubMedCrossRefGoogle Scholar
  10. 10.
    Garcia-Perez J, Sanchez-Palomino S, Perez-Olmeda M, et al. A new strategy based on recombinant viruses as a tool for assessing drug susceptibility of human immunodeficiency virus type 1. J Med Virol 2007; 79: 127–37PubMedCrossRefGoogle Scholar
  11. 11.
    Derdeyn CA, Decker JM, Sfakianos JN, et al. Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J Virol 2000; 74: 8358–67PubMedCrossRefGoogle Scholar
  12. 12.
    Takeuchi Y, McClure MO, Pizzato M. Identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research. J Virol 2008; 82: 12585–8PubMedCrossRefGoogle Scholar
  13. 13.
    Wei X, Decker JM, Liu H, et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 2002; 46: 1896–905PubMedCrossRefGoogle Scholar
  14. 14.
    Wei X, Decker JM, Wang S, et al. Antibody neutralization and escape by HIV-1. Nature 2003; 422: 307–12PubMedCrossRefGoogle Scholar
  15. 15.
    Chong H, Hong K, Zhang C, et al. Genetic and neutralization properties of HIV-1 env clones from subtype B/BC/AE infections in China. J Acquir Immune Defic Syndr 2008; 47: 535–43PubMedCrossRefGoogle Scholar
  16. 16.
    Betts BJ, Shafer RW. Algorithm specification interface for human immunodeficiency virus type 1 genotypic interpretation. J Clin Microbiol 2003; 41: 2792–4PubMedCrossRefGoogle Scholar
  17. 17.
    Kantor R, Machekano R, Gonzales MJ, et al. Human immunodeficiency virus reverse transcriptase and protease sequence database: an expanded data model integrating natural language text and sequence analysis programs. Nucleic Acids Res 2001; 29: 296–9PubMedCrossRefGoogle Scholar
  18. 18.
    Koup RA, Ho DD, Poli G, et al. Isolation and quantitation of HIV in peripheral blood. Curr Protoc Immunol 2001; Chapter 12: Unit 12.2Google Scholar
  19. 19.
    Rhee SY, Taylor J, Wadhera G, et al. Genotypic predictors of human immunodeficiency virus type 1 drug resistance. Proc Natl Acad Sci U S A 2006; 103: 17355–60PubMedCrossRefGoogle Scholar
  20. 20.
    Ceccherini-Silberstein F, Svicher V, Sing T, et al. Characterization and structural analysis of novel mutations in human immunodeficiency virus type 1 reverse transcriptase involved in the regulation of resistance to nonnucleoside inhibitors. J Virol 2007; 81(20): 11507–19PubMedCrossRefGoogle Scholar
  21. 21.
    Girouard M, Diallo K, Marchand B, et al. Mutations E44D and V118I in the reverse transcriptase of HIV-1 play distinct mechanistic roles in dual resistance to AZT and 3TC. J Biol Chem 2003; 278: 34403–10PubMedCrossRefGoogle Scholar
  22. 22.
    Gianotti N, Galli L, Boeri E, et al. The 118I reverse transcriptase mutation is the only independent genotypic predictor of virologic failure to a stavudine-containing salvage therapy in HIV-1-infected patients. J Acquir Immune Defic Syndr 2006; 41: 447–52PubMedCrossRefGoogle Scholar
  23. 23.
    Deshpande A, Jauvin V, Magnin N, et al. Resistance mutations in subtype C HIV type 1 isolates from Indian patients of Mumbai receiving NRTIs plus NNRTIs and experiencing a treatment failure: resistance to AR. AIDS Res Hum Retroviruses 2007; 23: 335–40PubMedCrossRefGoogle Scholar
  24. 24.
    Ferradini L, Jeannin A, Pinoges L, et al. Scaling up of highly active antiretroviral therapy in a rural district of Malawi: an effectiveness assessment. Lancet 2006; 367: 1335–42PubMedCrossRefGoogle Scholar
  25. 25.
    Jackson JB, Becker-Pergola G, Guay LA, et al. Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission. AIDS 2000; 14: F111–5PubMedCrossRefGoogle Scholar
  26. 26.
    Jourdain G, Ngo-Giang-Huong N, Le CS, et al. Intrapartum exposure to nevirapine and subsequent maternal responses to nevirapine-based antiretroviral therapy. N Engl J Med 2004; 351: 229–40PubMedCrossRefGoogle Scholar
  27. 27.
    Kantor R, Katzenstein DA, Efron B, et al. Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration. PLoS Med 2005; 2: e1 12CrossRefGoogle Scholar
  28. 28.
    Kassaye S, Lee E, Kantor R, et al. Drug resistance in plasma and breast milk after single-dose nevirapine in subtype C HIV type 1: population and clonal sequence analysis. AIDS Res Hum Retroviruses 2007; 23: 1055–61PubMedCrossRefGoogle Scholar
  29. 29.
    Grossman Z, Istomin V, Averbuch D, et al. Genetic variation at NNRTI resistance-associated positions in patients infected with HIV-1 subtype C. AIDS 2004; 18: 909–15PubMedCrossRefGoogle Scholar
  30. 30.
    Whitcomb JM, Parkin NT, Chappey C, et al. Broad nucleoside reversetranscriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates. J Infect Dis 2003; 188: 992–1000PubMedCrossRefGoogle Scholar

Copyright information

© Adis Data Information BV 2011

Authors and Affiliations

  • Zheng Jia
    • 1
    • 2
  • Sihong Xu
    • 2
  • Jianhui Nie
    • 2
  • Jingyun Li
    • 3
  • Ping Zhong
    • 4
  • Wenbo Wang
    • 2
  • Youchun Wang
    • 1
    • 2
  1. 1.Chinese Academy of Medical Sciences & Peking Union Medical CollegeBeijingP.R. China
  2. 2.Department of Cell BiologyNational Institutes for Food and Drug ControlP.R. China
  3. 3.Center of AIDS, Institute of Microbiology and EpidemiologyAcademy of Military Medical ScienceBeijingP.R. China
  4. 4.Shanghai Center of Disease ControlShanghaiP.R. China

Personalised recommendations