These samples were tested for antibodies against a panel of 43 antigens

consisting of 18 peptides of the gp41 immunodominant region representing the majority of all known HIV/SIV lineages, including SIVwrc, and 25 peptides of the V3 region, including the four groups of HIV-1, HIV-2, SIVcol and representatives of the different SIVcpz/gor lineages that circulate among chimpanzees and gorillas in central Africa [17, 33]. To that end, we used a newly developed assay based on the Luminex technology. This new Luminex test is comparable to SIV specific ELISAs [[33, 43], Ayouba et al., manuscript in Akt inhibitor preparation]. This test is also an EIA, with the reaction support consisting of calibrated polystyrene beads on which peptides are covalently bound. Each peptide was immobilized on a distinct bead set with a unique LY294002 order fluorescence wavelength. Once covalently linked to bead, the 43 different peptides were mixed and distributed in wells of a semi-permeable ELISA plate like support. Diluted (100 μl, 1/200) antibodies-containing fluids

(serum, plasma or whole blood) were then added and incubated at room temperature for an hour with continuous shaking. After washing, 50 μl of a biotin-labelled anti-human IgG was added in each well and the plate was incubated 30 minutes at room temperature, while shaking. After a second series of washing, R-phycoerythrine-labelled streptavidine SB202190 mw was added for 10 minutes and washed out afterwards. The complex consisting of beads-peptide and the different additives was resuspended in buffer and read on a BioPlex-200 (BioRad,

Marnes la Coquette, France). With the Luminex mafosfamide technology, each bead set is sorted in a specific area of a 2 dimensional display, according to its wavelength of fluorescence, like in flow cytometry. For each sample and for each antigen, results are expressed as median fluorescence intensity per 100 beads. Cut-off values have been calculated for each of the 43 peptides from their reactivities against 95 SIV negative non-human primates’ samples and 50 HIV negative human plasma. Samples presenting MFI higher than 500 against a given were considered positive for that peptide. PCR analyses For PCRs the following tissues were used: spleen (n = 21), liver (n = 3), muscle (n = 2), heart and/or lung (n = 2), lymphnode (n = 1) and buffy-coat (n = 1). For 2 chimpanzees no material was available for PCR analyses. DNA was extracted with the DNA tissue (or blood) kit (Qiagen, Hilden, Germany). Samples were tested with a generic SIV PCR known to detect most primate Lentiviruses [44]. We used the primers DR1 (TRC AYA CAG GRG CWG AYG A) and DR2 (AIA DRT CAT CCA TRT AYT G) in the first round PCR and primers DR4 (GGI ATW CCI CAY CCD GCA GG) and DR5 (GGI GAY CCY TTC CAY CCY TGH GG) in a nested PCR. The cycling conditions were 94°C for 2 minutes, 30 × [94°C for 15 seconds, 50°C decreasing by 0.