Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIVRNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5–1 Gagpol mRNA+/Gag protein+ cells per million CD4 T cells. Uniquely, the HIVRNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.

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We thank J. Girouard, the clinical staff at McGill University Health Centre and all study participants; D. Gauchat, the CRCHUM Flow Cytometry Platform, O. Debbeche, the CRCHUM BSL3 Platform, D. Zenklusen and C. Lai for technical assistance; and D. Malayter for technical support. This study was supported by the National Institutes of Health (HL-092565, AI100663 CHAVI-ID, AI113096, AI118544), the Delaney AIDS Research Enterprise (DARE; 1U19AI096109), the Canadian Institutes for Health Research (137694; Canadian HIV Cure Enterprise), a Canada Foundation for Innovation grant, the FRQS AIDS and Infectious Diseases Network and the Foundation for AIDS Research (108928-56-RGRL). D.E.K. and N.C. are supported by FRQS Research Scholar Awards. A.F. is the recipient of a Canada Research Chair. J.-P.R. is the holder of the Louis Lowenstein Chair, McGill University. A.E.B. is the recipient of a CIHR Fellowship (award no. 152536). J.N. is the recipient of a scholarship from the Bavarian Research Alliance (BayFor). J.R. is the recipient of CIHR Fellowship Award no. 135349. N.A. is the recipient of a King Abdullah scholarship from the Saudi government.

Author information

Author notes

    • Filippos Porichis

    Current address: EMD Serono-Merck, Billerica, Massachusetts, USA.


  1. Research Centre of the Centre Hospitalier de l'Université de Montréal (CRCHUM) and Université de Montréal, Montreal, Quebec, Canada.

    • Amy E Baxter
    • , Julia Niessl
    • , Rémi Fromentin
    • , Jonathan Richard
    • , Marta Massanella
    • , Nathalie Brassard
    • , Nirmin Alsahafi
    • , Andrés Finzi
    • , Nicolas Chomont
    •  & Daniel E Kaufmann
  2. Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), La Jolla, California, USA.

    • Amy E Baxter
    • , Julia Niessl
    •  & Daniel E Kaufmann
  3. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA.

    • Filippos Porichis
  4. Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.

    • Nirmin Alsahafi
    •  & Andrés Finzi
  5. Chronic Viral Illnesses Service and Division of Hematology, McGill University Health Centre, Montreal, Quebec, Canada.

    • Jean-Pierre Routy


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A.E.B., F.P. and D.E.K. conceived and developed the HIVRNA/Gag assay, with input from A.F. and N.C.; A.E.B., J.N., R.F., J.R., N.B., M.M. and N.A. modified the protocol, designed specific experiments and provided reagents; J.-P.R. obtained IRB approval and recruited participants to provide primary samples; D.E.K. provided supervision; A.E.B. and D.E.K. wrote the manuscript and all authors approved the final version.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daniel E Kaufmann.

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