Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960

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 evolution(1,2). A viral sequence from 1959 ( ZR59) is the oldest known HIV- 1 infection(1). 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 Leopoldville, 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.