NHP Samples

Figure 1

Figure 1. Sites in the Democratic Republic of Congo where dried blood spots of nonhuman primates were collected (red circles).

For the validation of the HIV/SIV xMAP assay, we used 142 well-characterized samples from our NHP reference panel in which SIV infection was either confirmed or ruled out by highly sensitive PCR approaches, and for which sufficient plasma was available (14,15). The panel included 93 SIV-negative samples from 8 species and 49 SIV-positive samples from 9 species (Table 1). For SIV prevalence studies, 330 samples were collected during May 2009–2010 as dried blood spots (DBSs) around 3 rural cities in DRC (Figure 1). Whole blood, collected from primate bushmeat, was spotted onto a filter 903 FTA card (Whatman Plc, Kent, UK). After air-drying at ambient temperature, DBSs were stored into individual envelopes at ambient temperature. Animals died 6–78 hours before sampling. All NHP samples were obtained with approval from the Ministry of Environment and Health and the National Ethics Committee. Similar to our previous studies, bushmeat samples were obtained through a strategy specifically designed not to increase demand (8,15).

Screening for Cross-Reactive HIV/SIV Antibodies

All DBS samples were screened with the new HIV/SIV multiplex microbead immunoassay technology, i.e., xMAP. Similarly as for the SIV ELISAs, we used peptides covering the immunodominant region of the gp41 transmembrane and V3-loop region from all major SIV/HIV lineages known at the time we conducted this study (Table 2). To avoid interpeptide and intrapeptide cross-linking, the 2 cysteins of the gp41 peptides were cyclicized during synthesis. For SIVcol, gp41 peptides could not be synthesized because of their low solubility; the V3-loop peptide was used to identify corresponding antibodies (14,15). Peptides were covalently coupled on carboxyl-functionalized fluorescent polystyrene beads (Luminex Inc.) by using the Bio-Plex Amine Coupling Kit (Bio-Rad Laboratories, Marnes-la-Coquette, France) according to the manufacturer’s instructions. Unreacted sites were blocked with blocking buffer from the Amine Coupling Kit (Bio-Rad Laboratories). Peptide-coupled microsphere preparations were counted by using a hemocytometer and stored in the dark at 4°C. Before use, peptide-coupled beads were vortexed (30 s), sonicated (30 s), and diluted to 4,000 beads/µL. Dilution and washing buffer consisted of phosphate-buffered saline (PBS) containing 0.75 mol/L NaCl, 1% (wt/vol) bovine serum albumin (Sigma Aldrich, St. Quentin Fallavier, France), 5% (vol/vol) fetal bovine serum (Gibco-Invitrogen, Cergy Pontoise, France), and 0.05% (vol/vol) Tween-20 (Sigma-Aldrich).

Assays were performed in 96 well flat-bottomed filter plates (Millipore, Tullagreen, Ireland). Plates were prewet with 100 µL assay buffer; 50 μL of bead mixture was subsequently added to each well. Liquid was aspirated with a vacuum manifold and wells were washed with 100 μL of assay buffer. Wells were then incubated with 100 μL plasma (diluted 1:200) for 60 min at room temperature in the dark on a plate shaker (300 rpm/min). For DBSs, 100 μL of eluate, obtained after overnight incubation of two 6-mm disks in 1 mL of hypertonic PBS, was added to peptide-coated beads. After washing, plates were incubated with 50 μL/well of 4 μg/mL biotinylated mouse antihuman IgG (BD Pharmingen, Le Pont de Claix, France) for 30 min at room temperature in the dark under continuous shaking. After washing, wells were incubated with 50 μL of 1 μg/mL streptavidin-R-phycoerythrin conjugate/well (Invitrogen/Molecular Probes, Cergy Pontoise, France) for 10 min in the dark while shaking. After 2 final washes with reading buffer (PBS containing 1% [wt/vol] bovine serum albumin), beads were resuspended in 125 µL reading buffer/well and analyzed by using BioPlex-200 (Bio-Rad Laboratories).

Data were analyzed by using BioPlex Software Manager version 5.0 (Bio-Rad Laboratories). For each bead set, >100 events were read and results were expressed as median fluorescence intensity (MFI) per 100 beads. The cutoff value was calculated for each peptide as the mean MFI for all antibody-negative reference serum samples plus 5 SD as an adaptation of the strategy defined for ELISA and was set at 200 MFI corresponding to a consensus value for all peptides (19). Sensitivity and specificity were calculated for homologous (same species) and heterologous (different species) antibody detection.

DBS samples were also tested for HIV cross-reactive antibodies by using the INNO-LIA HIV Confirmation Test (Innogenetics, Gent, Belgium) as described (10). This test configuration includes HIV-1 and HIV-2 recombinant proteins and synthetic peptides that are coated as discrete lines on a nylon strip.

DNA Extraction and NHP Species Confirmation

Total DNA was extracted from all DBSs by using the Nuclisens MiniMAG Extraction Kit (Biomerieux, Craponne, France) according to the manufacturer’s instructions. Minor changes consisted of increasing the incubation time (2 h) of the viral lysis step to increase DNA release (20). Species identification recorded in the field was confirmed on all samples by amplifying a 386-bp mitochondrial DNA fragment of the 12S rRNA gene with primers 12S-L1091 and 12S-H1478 (21). PCR products were purified by electrophoresis on a 1% agarose gel and directly sequenced (ABI PRISM Big Dye Terminator Cycle sequencing Ready Reaction Kit with amplitaq FS DNA polymerase) on an automated sequencer (ABI 3130XL, Applied Biosystems, Courtaboeuf, France). Sequences were then assembled by using the software package Lasergene (DNASTAR, Inc, Madison, WI, USA).

Molecular Characterization and Phylogenetic Analyses of SIVs

PCR analyses were performed on SIV antibody-positive samples by using described conditions with universal HIV/SIV and SIV lineage-specific primers in pol or env shown in Table 3 (8,15,22–24). PCR products were purified by electrophoresis on a 1% agarose gel and directly sequenced as described above. Newly derived SIV nucleotide sequences were aligned with reference sequences of the different HIV/SIV lineages with MEGA4 and ClustalX version 2 (25) and minor manual adjustments when necessary. Nucleotide sites that could not be unambiguously aligned were excluded from the analyses. Appropriate models of evolution were selected for each data set by using Topali software (26), and maximum-likelihood phylogenies were reconstructed by using PhyML (27). The analyses were performed by using discrete gamma distribution and generalized time-reversible model. The starting tree was obtained by using PhyML. One hundred bootstrap replications were performed to assess confidence in topology. New sequences have been deposited in GenBank under accession nos. JN020273–JN020279 and GU989632.

Performance of the HIV/SIV Lineage Specific xMAP Assay on a Reference Panel of NHP Samples

Table 4 summarizes the sensitivity and specificity of homologous and heterologous antibody detection of the xMAP assay on the same reference panel that was used for the SIV lineage–specific ELISAs (17,18). The homologous gp41 peptide was available for 39 samples; 34 (87.2%) reacted with their gp41 peptide counterpart. Similarly, 46 (93.9%) of the 49 samples for which the homologous V3 peptide was available reacted with their V3 peptide counterpart. The combination of homologous gp41 and V3 peptides identified 47 (95.9%) of the 49 SIV-positive samples. All 49 SIV positive samples were identified by combining homologous and heterologous gp41 reactivities, including SIVcol positive samples for which no homologous gp41peptide was available, resulting in 100% sensitivity. The 3 SIVmnd samples that were not detected by the homologous gp41 peptide were all detected with the SIVmnd V3 peptide, and the SIVcpzPtt sample that was not detected by the SIVcpz peptides was reactive with the HIV-1 N gp41 peptide. Each gp41 peptide cross-reacted with >1 sample from a different primate species (data not shown); highest cross-reactivities were for SIVmus (23/48, 47.9%) and SIVsmm (30/48, 62.5%) peptides. Finally, none of the negative serum samples showed positive results with homologous gp41 or V3 peptides. However, 2 (1 Cercopithecus nictitans and 1 C. cephus monkeys) reacted weakly (MFI/cutoff ratio <2) with a single heterologous V3 peptide from SIVagm, resulting in an overall 100% and 97.9% specificity of homologous and heterologous antibody detection, respectively. Given the extraordinary SIV diversity, few false-negative samples were observed, and the combination of all peptides in a single well resulted in 100% sensitivity and 97.5% specificity. Thus, the new assay should enable detection of most SIV infections.

NHP Species Collected as Bushmeat at the Different Localities in DRC

DBS samples were obtained from 330 NHPs in 3 sites, but most (258/330, 78.2%) were collected around Kole (Figure 1). Species were identified in the field by pictographs and confirmed by sequence analysis of the 12S rRNA gene. This analysis identified 7 species: 147 yellow-nosed red-tailed guenons (C. ascanius whitesidei), 79 Tshuapa red colobus monkeys (Piliocolobus tholloni), 33 Wolf’s monkeys (C. mona wolfi), 33 black mangabeys (Lophocebus atterrimus atterrimus), 25 Angolan pied colobus (Colobus angolensis angolensis), 10 De Brazza monkeys (C. neglectus), and 3 Allen swamp monkeys (Allenopithecus negroviridis) (28). Four of the 7 species or subspecies are only present in DRC, i.e., red-tailed guenons, tshuapa red colobus, black mangabeys, and Wolf’s monkeys (28).

Prevalence and Genetic Diversity of SIVs in DRC

Because 4 of the 7 NHP species or subspecies are only present in DRC, they are most likely infected with SIVs that have not been documented, and antibody detection will thus depend on extent of cross-reactivity with antigens of known HIV/SIV lineages. Therefore, we screened all samples with the new SIV/HIV xMAP assay and with the HIV line immune assay (INNO-LIA) confirmation assay, which we previously used to detect a large diversity of SIV infections (8). Table 5 shows the number of SIV-positive and indeterminate samples per species. SIV infection was documented in 6 species and the overall prevalence was 19% (64/330) ranging from 0% to 25% per species. Highest SIV prevalences were seen in red-tailed guenons (25%) and Tshuapa red colobus (24%), which represent 70% (226/330) of the primate bushmeat collected in this study. We also observed HIV/SIV cross-reactive antibodies in De Brazza monkeys (20%), Wolf’s monkeys (12 %), black mangabeys (3%), and Angola pied colobus (4%). In addition, 3% (10/330) of the samples were considered as indeterminate for SIV. Notably, all samples from Tsuapa red colobus were only reactive in the xMAP assay and showed strong cross-reactivity with SIVwrc antigens from western red colobus (Piliocolobus badius), illustrating clearly the need for including a wide variety of SIV antigens to uncover new SIV lineages.

Genetic Diversity of SIVS in DRC

Figure 2

Figure 2. Phylogenetic relationships of the newly derived simian immunodeficiency virus (SIV) sequences in pol to representatives of the other SIV lineages. Newly identified strains in this study are in red and reference...

To confirm SIV infection and document SIV diversity, all SIV-positive and indeterminate samples were subjected to PCR amplification. Although DNA integrity was sufficient to confirm the primate species in all DBSs, proviral SIV DNA could only be amplified in pol (400 bp) for 8 samples, most likely because of DNA degradation related to long and suboptimal storage at ambient temperature in the field and the fact that animals died several days before sampling. SIV infection was confirmed in 4 red-tailed guenons, 1 Wolf’s monkey, 1 De Brazza monkey, and 2 Tshuapa red colobus. Phylogenetic tree analysis shows the presence of new SIV lineages in Wolf‘s monkeys and Tshuapa red colobus (Figure 2). SIVwol is close to SIVden obtained from Dent’s monkeys (C. mona denti), which are found in eastern DRC but without overlapping habitats with Wolf’s monkeys in central DRC (28). SIVtrc from Tshuapa red colobus forms a separate lineage although related to SIVkrc from Kibale red colobus in eastern Africa (29). SIVasc from red-tailed guenons forms a species-specific lineage with SIVasc described in a capitive animal, but a high genetic diversity is seen (30). The reported pol sequence from a captive black mangabey housed in the zoo in Kinshasa, also falls within the SIVasc radiation (31). Finally, SIVdeb clustered within the species-specific SIVdeb lineage observed for De Brazza monkeys across central Africa (32).