Identification of a new simian immunodeficiency virus lineage with a vpu gene present among different cercopithecus monkeys (C. mona, C. cephus, and C. nictitans) from Cameroon

Abstract

During a large serosurvey of wild-caught primates from Cameroon, we found 2 mona monkeys (Cercopithecus mona) out of 8 and 47 mustached monkeys (Cercopithecus cephus) out of 302 with human immunodeficiency virus (HIV)-simian immunodeficiency virus (SIV) cross-reactive antibodies. In this report, we describe the full-length genome sequences of two novel SIVs, designated SIVmon-99CMCML1 and SIVmus-01CM1085, isolated from one mona (CML1) and one mustached (1085) monkey, respectively. Interestingly, these viruses displayed the same genetic organization (i.e., presence of a vpu homologue) as members of the SIVcpz-HIV type 1 lineage and SIVgsn isolated from greater spot-nosed monkeys (Cercopithecus nictitans). Phylogenetic analyses of SIVmon and SIVmus revealed that these viruses were genetically distinct from other known primate lentiviruses but were more closely related to SIVgsn all across their genomes, thus forming a monophyletic lineage within the primate lentivirus family, which we designated the SIVgsn lineage. Interestingly, mona, mustached, and greater spot-nosed monkeys are phylogenetically related species belonging to three different groups of the genus Cercopithecus, the C. mona, C. cephus, and Cercopithecus mitis groups, respectively. The presence of new viruses closely related to SIVgsn in two other species reinforces the hypothesis that a recombination event between ancestral SIVs from the family Cercopithecinae is the origin of the present SIVcpz that is widespread among the chimpanzee population.

FIG. 1.

Antibody profiles observed in C. mona and C. cephus using a line immunoassay (INNO-LIA HIV Confirmation). The five HIV-1 antigens are synthetic peptides for the exterior envelope glycoprotein (sgp120), as well as recombinant proteins for the transmembrane envelope glycoprotein (gp41) and integrase (p31), core (p24), and matrix (p17) proteins. The HIV-2 antigens are synthetic peptides for the exterior envelope glycoprotein (sgp105), as well as recombinant gp36 protein. Plasma samples which recognized at least one HIV antigen with an intensity equal to or greater than the assay ± cutoff line were scored as positive, samples which exhibited weaker but still visible reactivities with at least two HIV antigens were scored as indeterminant, and samples which yielded no reactivity or only a single band of less than plus-minus intensity were scored as negative. The 3+, 1+, and ± bands evident in the top portions of all test strips control for sample addition (the presence of plasma immunoglobulin) and test performance (binding of secondary antibody).

FIG. 2.

Alignment of Vpu amino acid sequences of SIVmon-99CMCML1, SIVmus-01CM1085, SIVgsn-99CM71, SIVgsn-99CM166, SIVcpzANT, SIVcpzTAN1, SIVcpzGAB, SIVcpzCAM5, SIVcpzUS, HIV1-YBF30, HIV1-MVP5180, and HIV1-MD. Identical residues are in solid boxes. Similar amino acids are in shaded boxes. The dashes indicate gaps introduced to optimize alignment.

FIG. 3.

Amino acid alignment of the V3 loop region of gp120 from SIVmon-99CMCML1, SIVmus-01CM1085, and representatives of all SIV-HIV lineages. The crown region is boxed. The dashes indicate gaps introduced to optimize alignment, and dots indicate identical residues.

FIG. 4.

Diversity plot analysis comparing SIVmus-01CM1085 with SIVmon-99CMCML1 and representatives of the six major lineages of the primate lentiviruses, i.e., SIVcpz, SIVmnd, SIVsyk, SIVsm, SIVagm, and SIVcol plus SIVgsn. The protein sequence differences are plotted for windows of 200 aa moved in steps of 10 aa.

FIG. 5.

Bootscanning of SIVmon-99CMCML1 (A) and SIVmus-01CM1085 (B) against representatives of the six major SIV lineages. Bootscan analysis was performed on the concatenated nucleotide alignment with the nonoverlapping regions of gag, pol, env, vif, and nef. Since saturation always occurred at the third codon position for all genes, the bootscan analysis was performed on first and second codon positions, with a sliding window of 500 nt with a 20-nt step increment and 1,000 bootstrap replicates. Below the graph, the bar shows the locations of the gene regions.

FIG. 6.

Unrooted maximum-likelihood trees of the pol (A) and env (B) genes, including 26 SIV-HIV strains of the six major SIV lineages, as well as other SIVs for which full-length sequences are available (SIVrcm, SIVmnd2, and SIVgsn) and the new SIVmon-99CMCML1 and SIVmus-01CM1085 isolates. The pol and env trees were inferred using only first and second codon positions, with the TrN + Inv + Γ (α = 1.3854; I = 0.1862) and the HKY + Inv + Γ (α = 1.1796; I = 0.2508; Ti/Tv = 0.9724) model of nucleotide substitution, respectively. Horizontal branch lengths are drawn to scale, with the bar representing 0.1 nucleotide replacements per site. The neighbor-joining method gives a similar tree topology. An asterisk along a branch indicates that the branch has a P value of <0.001 in the maximum-likelihood analysis and a percentage of bootstrap replicates (out of 1,000) of >70 in the neighbor-joining tree. α, shape parameter of the Γ distribution; I, proportion of invariable sites; Ti/Tv, expected transition/transversion ratio.

FIG. 7.

Diversity plot analysis comparing SIVmus-SIVmon, SIVmus-SIVgsn, and SIVmus-SIVgsn with SIVagmGRI-SIVagmVER, SIVmnd1-SIVlhoest, SIVsun-SIVlhoest, and SIVcpzGAB-SIVcpzANT. The protein sequence difference is plotted for windows of 200 aa moved in steps of 10 aa.

FIG. 8.

Diversity plots of concatenated nucleotide alignments with the nonoverlapping regions of gag, pol, env, vif, and nef comparing SIVcpz from P. t. schweinfurthii (SIVcpzANT and SIVcpzTAN1) and SIVcpz from P. t. troglodytes (SIVcpzUS and SIVcpzGAB1) to SIVmus-01CM1085, SIVgsn-99CM166, SIVmon-99CMCML1, SIVsyk, SIVrcmNG411, and SIVmnd2. Simplot analysis was performed on first and second codon positions with a sliding window of 450 nt moved in steps of 20 nt.

FIG. 9.

Map of Africa showing the natural ranges of mona monkeys (C. mona), mustached monkeys (C. cephus), and greater spot-nosed monkeys (C. nictitans).