Human immunodeficiency virus type 1 (HIV-1) sequences that pre-date the recognition of AIDS are critical to defining the time of origin and the timescale of virus evolution1,2. A viral sequence from 1959 (ZR59) is the oldest known HIV-1 infection1. Other historically documented sequences, important calibration points to convert evolutionary distance into time, are lacking, however; ZR59 is the only one sampled before 1976. Here we report the amplification and characterization of viral sequences from a Bouin’s-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Léopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo (DRC)), and we use them to conduct the first comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1 group M viruses. Phylogenetic analyses position this viral sequence (DRC60) closest to the ancestral node of subtype A (excluding A2). Relaxed molecular clock analyses incorporating DRC60 and ZR59 date the most recent common ancestor of the M group to near the beginning of the twentieth century. The sizeable genetic distance between DRC60 and ZR59 directly demonstrates that diversification of HIV-1 in west-central Africa occurred long before the recognized AIDS pandemic. The recovery of viral gene sequences from decades-old paraffin-embedded tissues opens the door to a detailed palaeovirological investigation of the evolutionary history of HIV-1 that is not accessible by other methods.

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Data deposits

The sequences reported in this study have been deposited in GenBank under accession numbers EU580739EU580854 and EU589211EU589236.


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We thank J. Wertheim and M. Sanderson for computational assistance, and L. Jewel for providing the Canadian control specimen. The NIH/NIAID and the David and Lucile Packard Foundation funded the research.

Author Contributions M.W., D.E.T., S.M.W. and M.T.P.G. designed the study. M.G., T.H., K.K. and M.T.P.G. performed digestion and extraction, PCR, quantitative PCR, cloning and sequencing experiments. M.T.P.G., M.G. and M.B. optimized DNA/RNA isolation methods and designed PCR assays. D.E.T., J.-J.M., E.V.M., J.-M.M.K. and R.M.K. organized and provided samples. M.W. analysed the data, performed the phylogenetic analyses, and wrote the paper. S.M.W. contributed to the analyses and writing. All authors discussed the results and commented on the manuscript.

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    • M. Thomas P. Gilbert

    Present address: Centre for Ancient Genetics, Biological Institute, University of Copenhagen, Copenhagen DK-2100, Denmark.


  1. Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA

    • Michael Worobey
    • , Marlea Gemmel
    • , Tamara Haselkorn
    •  & M. Thomas P. Gilbert
  2. Sanofi Pasteur, F-69367 Lyon Cedex 07, France

    • Dirk E. Teuwen
  3. UCB SA Pharma, Braine l’Alleud, BE-1420, Belgium

    • Dirk E. Teuwen
  4. The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA

    • Kevin Kunstman
    •  & Steven M. Wolinsky
  5. Ancient DNA Laboratory, School of Biological Sciences and Biotechnology, Murdoch University, Perth, Western Australia 6150, Australia

    • Michael Bunce
  6. Department of Anatomy and Pathology, University of Kinshasa, Kinshasa B.P. 864, Democratic Republic of the Congo

    • Jean-Jacques Muyembe
    • , Jean-Marie M. Kabongo
    •  & Raphaël M. Kalengayi
  7. National Institute for Biomedical Research, National Laboratory of Public Health, Kinshasa B.P. 1197, Democratic Republic of the Congo

    • Jean-Jacques Muyembe
  8. Department of Pathology, University Hospital, University of Antwerp, Antwerp B-2610, Belgium

    • Eric Van Marck


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Corresponding author

Correspondence to Michael Worobey.

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