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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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.
This file contains Authenticity of Data, Supplementary Table S1 and Supplementary Figures S1-S3 with Legends