Timing of the HIV-1 subtype C epidemic in Ethiopia based on early virus strains and subsequent virus diversi®cation Almaz Abebe, Vladimir V. Lukashov a , Georgios Pollakis a , Aletta Kliphuis a , Arnaud L. Fontanet b , Jaap Goudsmit a and Tobias F. Rinke de Wit Objective: To trace the introduction of HIV-1 subtype C into Ethiopia based on virus diversi®cation during the epidemic. Design: A set of 474 serum samples obtained in Ethiopia in 1982±1985 was tested for HIV-1. HIV-1 env gp120 V3 and gag or pol regions were sequenced and analysed together with sequences from later stages of the epidemic. Results: None of 98 samples from 1982±1983, one of 193 samples from 1984, and one of 183 samples from 1985 were HIV-1 positive. Phylogenetic analysis of virus sequences from positive samples revealed that they belong to the Ethiopian C, and not the C9, cluster. Analysis of 81 Ethiopian C V3 sequences from 1984±1997 revealed that the consensus sequence of the Ethiopian epidemic has been stable over time. Both the 1984 and 1985 V3 sequences, in contrast with three out of 27 (11%) of the 1988 and none out of 51 of the 1992±1997 sequences, had no synonymous substitu- tions compared to the reconstructed common ancestor of the Ethiopian C viruses. A highly signi®cant correlation between sampling years of the V3 sequences and their synonymous distances to the common ancestor was demonstrated. Conclusions: The increasing genetic heterogeneity together with stable consensus sequence of the Ethiopian HIV-1 C population demonstrates that evolution of the virus population is characterized by an unbiased expansion around a stationary consensus. Based on the rate of synonymous diversi®cation of HIV-1 strains within the Ethiopian population, we were able to estimate 1983 (95% con®dence interval, 1980±1984) as the year of HIV-1 C introduction into Ethiopia. & 2001 Lippincott Williams & Wilkins AIDS 2001, 15:1555±1561 Keywords: Ethiopia, HIV-1 epidemic, HIV-1 genetic characterization, molecular clock, subtype C Introduction Like most countries in Sub-Saharan Africa, Ethiopia has experienced a severe HIV-1/AIDS epidemic during the last 15 years. Intensive retrospective seroepidemio- logical studies, the results of which are summarized in Table 1, have revealed the absence of HIV-1 in rural or urban Ethiopian populations prior to 1984 [1,2]. From the Ethiopian±Netherlands AIDS Research Project (ENARP) at the Ethiopian Health and Nutrition Research Institute (EHNRI), Addis Ababa, Ethiopia, the a Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands, and the b Division of public Health and Environment, Municipal Health Service, Amsterdam, the Netherlands. Note: Almaz Abebe and Vladimir V. Lukashov contributed equally to this work. Requests for reprints to: A. Abebe, Ethiopian Health and Nutrition Research Institute, PO box 1242, Addis Ababa, Ethiopia. Received: 23 May 2000; revised: 6 November 2000; accepted: 24 April 2001. ISSN 0269-9370 & 2001 Lippincott Williams & Wilkins 1555