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Detection and molecular characterization of SIVcpz in chimpanzee communities in the vicinity of Kisangani should directly test the OPV/AIDS theory. An earlier survey of chimpanzees at Wanie-Rukula near Kisangani (Fig. 1a; W. D. Hamilton, M.W. and J. B. J., January 2000, see ref. 9) failed to identify SIVcpz viral (v) RNA in any of 34 faecal samples collected. However, western immunoblot analysis of 10 chimpanzee urine samples collected at the same time identified two specimens that showed strong crossreactivity with the HIV-1 core protein p24 (Fig. 1b). Such indeterminate urine antibody profiles were found in chimpanzees from Tanzania, where SIVcpz infection was subsequently demonstrated after amplification by polymerase chain reaction (PCR) and sequencing of DNA10.

Figure 1: The Kisangani expeditions and refutation of the OPV/AIDS theory.
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a, Map of the Democratic Republic of Congo (DRC) and neighbouring countries showing the ranges of Pan troglodytes troglodytes (Ptt) and Pan troglodytes schweinfurthii (Pts). b, Western blot analysis of urine samples from 10 chimpanzees, collected during the 2000 expedition to rainforests near Kisangani; plasma from a positive HIV-1 control is shown in the left lane, with crossreactivity to the different viral proteins indicated. Two samples showed strong crossreactivity with HIV-1 p24 (arrowhead). c, Maximum-likelihood-estimated phylogenetic tree for gp41/nef sequence data, with bootstrap results. Bootstrap percentages are shown for all clades that were present in more than 50% of 1,000 maximum-likelihood-inferred bootstrap trees. Support for the SIVcpzDRC1/SIVcpzTAN clade was very strong (99%). Analysis of partial gag sequences supported the same phylogenetic position for SIVcpzDRC1 (data not shown). For further details of phylogenetic analyses, see supplementary information.

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To confirm the existence of SIVcpz in the Kisangani apes and to identify circulating strain(s) at a molecular level, we resumed field-work in February 2003, this time collecting 97 faecal samples from three different sites (for map, see supplementary information). From these, we identified one SIVcpz vRNA-positive specimen from the Parisi forest by PCR amplification of gag (422 base pairs) and gp41/nef (699 base pairs) sequences. This result confirmed that natural SIVcpz infection was present in chimpanzees in the Kisangani region.

Phylogenetic analysis of the newly derived sequences revealed that the Kisangani virus clustered with high statistical support with SIVcpz strains that were infecting chimpanzees of the same subspecies (Pan troglodytes schweinfurthii) that lived about 800 km to the south-east in Gombe National Park in Tanzania10,11. The new virus, which we designate SIVcpzDRC1, represents a third lineage within the well circumscribed P. t. schweinfurthii SIVcpz radiation, and is clearly distinct from the P. t. troglodytes SIVcpz clade that includes all known strains of HIV-1 (Fig. 1c, and see supplementary information).

These results indicate that chimpanzees in the vicinity of Kisangani are endemically infected with SIVcpz that is highly divergent from HIV-1, thereby ruling out these apes as the source of HIV-1 and refuting the OPV/AIDS theory. Instead, each of the many circulating HIV-1 variants comprising groups M, N and O is linked to SIVcpz from P. t. troglodytes (Fig. 1c), the chimpanzee subspecies native to west-central Africa1,12.

Given that fears about the safety of polio vaccines are currently threatening the global campaign to eradicate the disease8, our clear-cut evidence against one of the key sources of concern is timely. The molecular epidemiological data presented here, together with data suggesting that HIV-1 group M originated 30 years before OPV trials were conducted1,13 and the absence of detectable SIVcpz or chimpanzee DNA in archival stocks of OPV2,3, should finally lay the OPV/AIDS theory to rest.