Full-length sequence analysis of SIVmus in wild populations of mustached monkeys (Cercopithecus cephus) from Cameroon provides evidence for two co-circulating SIVmus lineages

Abstract

Mustached monkeys (Cercopithecus cephus), which form a significant component of primate bushmeat in west central Africa, are infected with simian immunodeficiency virus (SIVmus). We identified and genetically characterized five new SIVmus strains infecting wild living mustached monkeys from Cameroon. Phylogenetic analysis of partial pol sequences revealed that SIVmus strains form two distinct groups within the clade comprised of lentiviruses isolated from Cercopithecus nictitans (SIVgsn), Cercopithecus mona (SIVmon) and C. cephus (SIVmus). Characterisation of three full-length SIVmus genomes confirmed the presence of two distinct lineages infecting mustached monkeys. These two variants of SIVmus, here designated SIVmus-1 and SIVmus-2, were isolated from animals sharing habitats within the same geographic region. Phylogenetic analyses showed that the diversification of SIVmus, SIVgsn and SIVmon involved inter-lineage recombination, and suggested that one of the SIVmus lineages likely resulted from cross-species transmission and recombination involving SIVmus and an as yet uncharacterized SIV. These results indicate that cross-species transmission and recombination play a major role in the evolution of primate lentiviruses among sympatric primate species.

Figure 1

Phylogenetic relationships of the newly derived SIVmus sequences in Pol (A) and Env (B) to representatives of the other SIV lineages. The unrooted trees were inferred from 609 amino acids (Pol) and 536 amino acids (Env). SIVmus sequences are highlighted. The numbers at nodes are posterior probabilities estimated by the Bayesian method. Only those 95% and above are shown. Scale bars represent 0.2 and 0.5 replacements per site.

Figure 2

Geographic origins of the SIVmus positive samples and range of C. cephus (in yellow), C. nictitans (in pink) and C. mona (in blue) in Cameroon. Within the area shown, the ranges of C. cephus and C. mona are overlapped by C. nictitans. The previously reported SIVmus-01CM1085 and new SIVmus positive samples from this study were collected in different villages, corresponding to two sampling sites covering a 25 km² area each, and separated by 40 km around the Dja reserve in south Cameroon. The sites where the previously characterized SIVgsn (99CM71 and 99CM166) and SIVmon (99CMCML1) samples were collected are also indicated on the map.

Figure 3

Amplification of full-length genomes of SIVmus: (A) unintegrated circular DNA was targeted for SIVmus-01CM1246 amplification, (B) linear overlapping fragments were amplified to generate the complete genomes for SIVmus-01CM1239 and SIVmus-01CM2500. Primers used to amplify the different PCR fragments are listed in Table 2.

Figure 4

Diversity plots of concatenated Gag, Pol, Vif, Env and Nef protein sequences showing distance from SIVmus-01CM1239 (A) and SIVmus-01CM2500 (B) to other viruses of the SIVgsn/mus/mon lineage. The proportion of amino acid sequence difference per 300 residues window (vertical axis) is plotted against the midpoint of the sequence window (horizontal axis).

Figure 5

Phylogenetic relationships of SIVmus, SIVgsn and SIVmon. Trees were inferred from protein sequence alignments from different parts of the genome; (A) Gag (sites 1-380 of the proteome alignment), (B) Pol1 (sites 381-896), (C) Pol2 (sites 897-1281) and (D) Env (sites 1415-2057). SIVsyk, SIVdeb and SIVden were used as outgroups to root the trees. Newly characterized full-length genomes are boxed and SIVmus groups are indicated. The numbers on internal branches are the estimated posterior probabilities from the Bayesian method (values of 95% and above are shown). Scale bars indicate 0.05 or 0.1 substitutions per site.