Simultaneous Ultrasensitive Subpopulation Staining/Hybridization In Situ (SUSHI) in HIV-1 Disease Monitoring
The field of virology is undergoing a revolution as diagnostic tests and new therapies are allowing clinicians to treat, monitor, and predict outcomes of viral diseases. The majority of these techniques, however, destroy the factory of viral production and the information inherent in the reservoir – the cell. In this chapter, we describe a technique that combines cell surface immunophenotyping (to unequivocally identify cell types) and ultrasensitive fluorescence in situ hybridization (U-FISH) for HIV-1 to detect productively infected cells. Identification of virus and host (cells) allows earlier detection of changes in viral production and viral suppression but most importantly allows clinicians to monitor response to anti-viral therapy on a cell-by-cell and tissue-by-tissue basis taking into account the fact that the human body consists of very different, distinct compartments with unique selection pressures exerted on the viral life cycle.
Key wordsVirus HIV Tropism T-cells Monocytes Flow cytometry Laser confocal image analysis Oligonucleotides
The author would like to acknowledge and thank Keith Shults for his collaborative efforts and unyielding support in the development of these techniques.
- 2.Furtado, M.R., Callaway, D.S., Phair, J.P., Kunstman, K.J., Stanton, J.L., Macken, C.A., Perelson, A.S., Wolinsky, S.M. (1999) Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy. N Engl J Med 340, 1614–1622PubMedCrossRefGoogle Scholar
- 3.Dronda, F., Moreno, S., Moreno A., Casado, JL., Perez-Elias, M.J., and Antela, A. (2002) Long-term outcomes among antiretroviral-naïve human immunodeficiency virus-infected patients with small increases in CD4+ counts after successful virologic suppression. Clin Infect Dis 35, 1005–1009.PubMedCrossRefGoogle Scholar
- 6.Li, S., Juarez, J., Alali, M., Dwyer, D., Collman, R., Cunningham, A., and Naif, H. (1999) Persistent CCR5 utilization and enhanced macrophage tropism by primary blood human immunodeficiency virus type 1 isolates from advanced stages of disease and comparison to tissue-derived isolates. J Virol 73, 9741–9755.PubMedGoogle Scholar
- 7.Kulkosky, J., Nunnari, G., Otero, M., Calarota, S., Dornadula, G., Zhang, H., Malin, A., Sullivan, J., Xu, Y., DeSimone, J., Babinchak, T., Stern, J., Cavert, W., Haase, A., Pomerantz, R. (2002) Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy. J Infect Dis 186, 1403–1411PubMedCrossRefGoogle Scholar
- 9.Folks T, Benn S, Rabson A, Theodore T, Hoggan MD, Martin M, Lightfoote M, Sell K. (1985). Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc Natl Acad Sci U S A 82, 4539–4543.PubMedCrossRefGoogle Scholar