Collection of Primate Tissue and Blood Samples

Blood was obtained from 788 monkeys wild-caught in Cameroon from January 1999 to April 2001. Species were determined by visual inspection according to the Kingdon Field Guide to African Mammals (13) and the taxonomy described by Colin Groves (14). We sampled 573 animals as bushmeat at markets in Yaounde (n=157), surrounding villages (n=111), or logging concessions in southeastern Cameroon (n=305), as well as 215 pet animals from these same areas (Table 1). All primate samples were obtained with government approval from the Cameroonian Ministry of Environment and Forestry. Bushmeat samples were obtained through a strategy specifically designed not to increase demand: women preparing and preserving the meat for subsequent sale and hunters already involved in the trade were asked for permission to sample blood and tissues from carcasses, which were then returned.

For the bushmeat animals, blood was collected by cardiac puncture, and lymph node and spleen tissues were collected whenever possible. The owners indicated that most of the animals had died 12 to 72 hours before sampling. For pet monkeys, blood was drawn by peripheral venipuncture after the animals were tranquilized with ketamine (10 mg/kg). Plasma and cells were separated on site by Ficoll gradient centrifugation. All samples, including peripheral blood mononuclear cells (PBMCs), plasma, whole blood, and other tissues, were stored at –20°C.

Serologic Testing

Plasma samples were tested for HIV/SIV antibodies by the INNO-LIA HIV Confirmation test (Innogenetics, Ghent, Belgium), which includes HIV-1 and HIV-2 recombinant proteins and synthetic peptides that are coated as discrete lines on a nylon strip. Five HIV-1 antigens include synthetic peptides for the exterior envelope glycoprotein (sgp120), as well as recombinant proteins for the transmembrane envelope glycoprotein (gp41), integrase (p31), core (p24), and matrix (p17) proteins. HIV-1 group O envelope peptides are included in the HIV-1 sgp120 band. The HIV-2 antigens include synthetic peptides for sgp120, as well as recombinant gp36 protein. In addition to these HIV antigens, each strip has control lines: one sample addition line (3+) containing anti-human immunoglobulin (Ig) and two test performance lines (1+ and +/-) containing human IgG. All assays were performed according to manufacturer’s instructions, with alkaline phosphatase-labeled goat anti-human IgG as the secondary antibody. We used the following working definition for SIV seropositivity: plasma samples were scored as INNO-LIA positive when they reacted with at least one HIV antigen and had a band intensity equal to or greater than the assay cutoff (+/-) lane; samples that reacted less strongly but still visibly with two or more HIV antigens were classified as indeterminant; and samples reacting with no bands or only one band with less than +/- intensity were classified as negative.

Polymerase Chain Reaction (PCR)

DNA was isolated from whole blood or PBMCs by using Qiagen DNA extraction kits (Qiagen, Courtaboeuf, France), and PCR was done with the Expand High Fidelity PCR kit (Roche Molecular Biochemicals, Mannheim, Germany). For amplification of SIV sequences, previously described degenerate consensus pol primers DR1, Polis4, UNIPOL2, and PolOR (15–17) were used in various combinations under previously described PCR conditions (16). PCR products were sequenced by cycle sequencing and dye terminator methods (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq FS DNA polymerase [PE Biosystems, Warrington, England]) on an automated sequencer (ABI 373, Stretch model; Applied Biosystems, Courtaboeuf, France) either directly or after cloning into the pGEM-T vector (Promega, Charbonnieres, France).

To test for DNA degradation, a 1,151-bp region of the glucose-6–phosphate dehydrogenase (G6PD) gene was amplified with the primers GPD-F1 5′-CATTACCAGCTCCATGACCAGGAC-3′ and GPD-R1 5′-GTGTTCCCAGGTGACCCTCTGGC-3′ in a single-round PCR reaction under the following conditions: 94°C for 2 min, then 35 cycles at 94°C for 20 sec; 58°C for 30 sec, and 72 °C for 1 min (18).

Phylogenetic Analyses

Newly derived SIV nucleotide sequences were aligned with reference sequences from the Los Alamos HIV/SIV Sequence database by using CLUSTAL W (19) with minor adjustments for protein sequences. A phylogenetic tree was constructed by the neighbor-joining method (20), and the reliability of branching orders was tested by the bootstrap approach (21). Sequence distances were calculated by Kimura’s two-parameter method (22). SIV lineages were defined as clusters of SIV sequences from the same primate species that grouped together with significant (>80%) bootstrap values.

GenBank Accession Numbers

The new sequences have been deposited in GenBank under the following accession numbers: SIVgsn-99CM-CN71 (AF478588), SIVgsn-99CM-CN7 (AF478589), SIVgsn-99CM-CN166 (AF478590), SIVmon-99CM-CML1 (AF478591), SIVmus-01CM-S1239 (AF478592), SIVmus-01CM-S1085 (AF478593), SIVtal-00CM-271 (AF478594), SIVtal-00CM-266 (AF478595), SIVmnd2-99CM-54 (AF478596), SIVmnd2-01CM-S109 (AF478597), SIVmnd2-00CM-S46 (AF478598), SIVmnd2-00CM-S6 (AF478599), SIVdeb-01CM-1083 (AF478600), SIVdeb-99CM-CN40 (AF478601), SIVdeb-01CM-S1014 (AF478602), SIVdeb-99CM-CNE5 (AF478603), SIVdeb-01CM-1161 (AF478604), SIVdeb-99CM-CNE1 (AF478605), SIVcol-00CM-247 (AF478606), SIVcol-00CM-243 (AF478607), and SIVcol-99CM-11 (AF478608).

Prevalence Estimates of SIV Infection in Bushmeat and Pet Monkey Samples

Previous studies of SIV infection have relied almost exclusively on surveys of captive monkeys or apes that were either kept as pets or housed at zoos, sanctuaries, or primate centers. While this approach has led to the discovery of novel SIVs (23–29), it has not provided information concerning SIV prevalence rates in the wild. Most pet monkeys are acquired at a very young age, often when their parents are killed by hunters. Two field studies of wild African green monkeys have shown that seroprevalence rates correlated with sexual maturity, suggesting transmission predominantly by sexual routes (30,31). SIV infection rates of captive monkeys may thus not accurately reflect SIV prevalence rates in the wild.

Figure 1

Figure 1. Detection of HIV-1/HIV-2 cross-reactive antibodies in sera from 11 primate species by using a line immunoassay (INNO-LIA HIV Confirmation, Innogenetics, Ghent, Belgium). Varying patterns of reactivity to HIV peptides and proteins...

To ensure systematic sampling, we therefore collected blood from 573 monkeys sold as bushmeat and 215 pet monkeys (Table 1). Most of the bushmeat animals were adults, while most of the pets were still infants or juveniles at the time of sampling. Most primates came from the southern part of the country. All major SIV lineages known to date were initially discovered because their primate hosts had antibodies that cross-reacted with HIV-1 or HIV-2 antigens (23–29). Although the extent of this cross-reactivity has not been defined, we used a similar approach to examine the primate blood samples obtained in Cameroon. Since commercially available HIV screening assays (e.g., enzyme-linked immunosorbent assay or rapid tests) contain only a limited number of antigens, we used an HIV confirmatory assay (INNO-LIA), comprising a recombinant and synthetic peptide-based line immunoassay (Figure 1). One hundred thirty-one (16.6%) of 788 plasma samples reacted strongly with one or more HIV antigens, while an additional 34 samples (4.3%) reacted less strongly but visibly with two or more HIV antigens (Figure 1; Table 2). Of 13 primate species that had HIV cross-reactive antibodies, the prevalence of seroreactivity (positive plus indeterminant) ranged from 5% to 40%. Prevalences were lower in pet animals than in bushmeat primates, 11.6% versus 18.4%, respectively. Sera from only three species failed to react completely (Cercopithecus preussi, Mandrillus leucophaeus, Cercocebus torquatus), but these three species accounted for only 5 of the 788 samples tested.

The INNO-LIA profiles from members of the same as well as different primate species varied extensively (Figure 1). Some sera reacted only with HIV core and/or Pol proteins, while others reacted with Gag and/or Pol and/or Env proteins from either HIV-1 or HIV-2 or both. Other than classifying sera as INNO-LIA reactive or nonreactive, no banding pattern or algorithm could be derived that would have been predictive of infection of any given primate species.

Confirmation of SIV Infection by PCR and Discovery of Novel SIV Lineages

Figure 2

Figure 2. Identification of diverse Simian immunodeficiency virus (SIV) lineages in primate bushmeat. A 650-bp pol fragment was amplified from monkeys representing seven primate species, sequenced, and subjected to phylogenetic tree analysis by...

A total of 342 samples, including INNO-LIA positive (n=91), indeterminant (n=23), or negative (n=228) specimens were subjected to PCR analysis (16,32), which yielded amplification products for 28 blood samples from seven primate species: Cercopithecus mona, C. neglectus, C. nictitans, C. cephus, Colobus guereza, Miopithecus ogouensis, and Mandrillus sphinx (Table 3). All these amplification products were of appropriate size. Moreover, subsequent sequence and phylogenetic analysis confirmed SIV infection (Figure 2). Most of the newly derived sequences did not fall into any of the known SIV groups. Viral sequences from C. mona (SIVmon), C. neglectus (SIVdeb), C. nictitans (SIVgsn), C. cephus (SIVmus), and Miopithecus ogouensis (SIVtal) formed species-specific monophyletic clusters that were roughly equidistant from each other as well as from all previously defined SIV lineages in this region of the pol gene. Viruses from the remaining two species (Colobus guereza and Mandrillus sphinx) grouped with previously reported SIVcol and SIVmnd-2 strains, respectively.

The single sequence of SIVmon was given lineage status because of its high degree of genetic diversity from the other SIV strains. We maintained the lineage designation of SIVtal previously assigned to a virus thought to be derived from a zoo animal of the species M. talapoin (28) because that sequence and the two newly derived talapoin viruses from M. ogouensis cluster together in a phylogenetic tree derived from additional pol nucleotide sequences (not shown). Thus, our new SIVtal sequences confirm the existence of this lineage in the wild .

SIV sequences were confirmed in 26 of 91 INNO-LIA-positive samples, as well as in 1 of 23 indeterminate and 1 of 223 negative samples (Table 3). Because many blood samples were obtained under poorly controlled circumstances, especially from the bushmeat markets, we tested the possibility of DNA degradation. Whole blood and PBMC DNA preparations were subjected to single-round PCR with primers designed to amplify introns 4 and 5 of the nuclear G6PD gene (1,100 bp). Of the 65 LIA-positive samples that did not yield a virus-specific PCR product, 11 also failed to yield a G6PD amplification product. Similarly, 4 of 17 INNO-LIA-indeterminate and SIV PCR-negative samples, as well as 25 of 102 INNO-LIA-negative samples, were also negative by G6PD amplification. These results indicate that, in addition to using only a single set of nested pol primer pairs, low PCR amplification rates from LIA-positive and -indeterminant samples were also due to DNA degradation, the presence of PCR inhibitors, or both.