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Journal of Clinical Microbiology, June 2006, p. 1930-1943, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02415-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evaluation of a Multiple-Cycle, Recombinant Virus, Growth Competition Assay That Uses Flow Cytometry To Measure Replication Efficiency of Human Immunodeficiency Virus Type 1 in Cell Culture

Carrie Dykes,1 Jiong Wang,1 Xia Jin,1 Vicente Planelles,2 Dong Sung An,3 Amanda Tallo,1 Yangxin Huang,4,{dagger} Hulin Wu,4 and Lisa M. Demeter1*

Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York,1 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah,2 Department of Hematology and Oncology, UCLA AIDS Institute, Los Angeles, California,3 Department of Biostatistics, University of Rochester School of Medicine and Dentistry, Rochester, New York4

Received 21 November 2005/ Returned for modification 20 January 2006/ Accepted 27 March 2006

Human immunodeficiency virus type 1 (HIV-1) replication efficiency or fitness, as measured in cell culture, has been postulated to correlate with clinical outcome of HIV infection, although this is still controversial. One limitation is the lack of high-throughput assays that can measure replication efficiency over multiple rounds of replication. We have developed a multiple-cycle growth competition assay to measure HIV-1 replication efficiency that uses flow cytometry to determine the relative proportions of test and reference viruses, each of which expresses a different reporter gene in place of nef. The reporter genes are expressed on the surface of infected cells and are detected by commercially available fluorescence-labeled antibodies. This method is less labor-intensive than those that require isolation and amplification of nucleic acids. The two reporter gene products are detected with similar specificity and sensitivity, and the proportion of infected cells in culture correlates with the amount of viral p24 antigen produced in the culture supernatant. HIV replication efficiencies of six different drug-resistant site-directed mutants were reproducibly quantified and were similar to those obtained with a growth competition assay in which the relative proportion of each variant was measured by sequence analysis, indicating that recombination between the pol and reporter genes was negligible. This assay also reproducibly quantified the relative fitness conferred by protease and reverse transcriptase sequences containing multiple drug resistance mutations, amplified from patient plasma. This flow cytometry-based growth competition assay offers advantages over current assays for HIV replication efficiency and should prove useful for the evaluation of patient samples in clinical trials.

* Corresponding author. Mailing address: University of Rochester School of Medicine and Dentistry, Infectious Diseases Unit, 601 Elmwood Ave., Box 689, Rochester, NY 14642. Phone: (585) 275-4764. Fax: (585) 442-9328. E-mail: lisa_demeter{at}urmc.rochester.edu.

{dagger} Present address: Department of Epidemiology and Biostatistics, College of Public Health, University of South Florida, Tampa, FL 33612.

Journal of Clinical Microbiology, June 2006, p. 1930-1943, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02415-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Boutwell, C. L., Rowley, C. F., Essex, M. (2009). Reduced Viral Replication Capacity of Human Immunodeficiency Virus Type 1 Subtype C Caused by Cytotoxic-T-Lymphocyte Escape Mutations in HLA-B57 Epitopes of Capsid Protein. J. Virol. 83: 2460-2468 [Abstract] [Full Text]  
  • Dykes, C., Demeter, L. M. (2007). Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness. Clin. Microbiol. Rev. 20: 550-578 [Abstract] [Full Text]  


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