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In contrast to humans, several primate species are believed to have harbored simian immunodeficiency viruses (SIVs) since ancient times. In particular, the geographically dispersed species of African green monkeys (AGMs) are all infected with highly diversified SIVagm viruses at high prevalences (greater than 50% of sexually mature individuals) without evident diseases, implying that the progenitor monkeys were infected prior to their dispersal. If this is correct, AGMs would be expected to have accumulated frequent resistance-conferring polymorphisms in host genes that are important for SIV replication. Accordingly, we analyzed the coding sequences of the CCR5 coreceptors from 26 AGMs (52 alleles) in distinct populations of the four species. These samples contained 29 nonsynonymous coding changes and only 15 synonymous nucleotide substitutions, implying intense functional selection. Moreover, 24 of the resulting amino acid substitutions were tightly clustered in the CCR5 amino terminus (D13N in the vervets and Y14N in the tantalus species) or in the first extracellular loop (Q93R and Q93K in all species). The Y14N substitution was extremely frequent in the 12 wild-born African tantalus, with 7 monkeys being homozygous for this substitution and 4 being heterozygous. Although two of these heterozygotes and the only wild-type homozygote were naturally infected with SIVagm, none of the Y14N homozygotes were naturally infected. A focal infectivity assay for SIVagm indicated that all five tested SIVagms efficiently use CCR5 as a coreceptor and that they also use CXCR6 (STRL33/Bonzo) and GPR15 (BOB) with lower efficiencies but not CXCR4. Interestingly, the D13N, Y14N, Q93R, and Q93K substitutions in AGM CCR5 all strongly inhibited infections by the SIVagm isolates in vitro. The Y14N substitution eliminates a tyrosine sulfation site that is important for infections and results in partial N-linked glycosylation (i.e., 60% efficiency) at this position. Nevertheless, the CCR5(Y14N) component that lacks an N-linked oligosaccharide binds the chemokine MIP-lbeta with a normal affinity and is fully active in signal transduction. Similarly, D13N and Q93R substitutions did not interfere with signal transduction. Thus, the common substitution polymorphisms in AGM CCR5 strongly inhibit SIVagm infections while substantially preserving chemokine signaling. In contrast, polymorphisms of human CCR5 are relatively infrequent, and the amino acid substitutions are randomly situated and generally without effects on coreceptor function. These results support an ancient coevolution of AGMs and SIVagm viruses and establish AGMs as a highly informative model for learning about host proteins that play critical roles in immunodeficiency virus infections.

Mandrillus sphinx, a large primate living in Cameroon and Gabon and belonging to the Papionini tribe, was reported to be infected by a simian immunodeficiency virus (SIV) (SIVmndGB1) as early as 1988. Here, we have identified a second, highly divergent SIVmnd (designated SIVmnd-2). Genomic organization differs between the two viral types; SIVmnd-2 has the additional vpx gene, like other SIVs naturally infecting the Papionini tribe (SIVsm and SIVrcm) and in contrast to the other SIVmnd type (here designated SIVmnd-1), which is more closely related to SIVs infecting l'hoest (Cercopithecus lhoesti lhoesti) and sun-tailed (Cercopithecus lhoesti solatus) monkeys. Importantly, our epidemiological studies indicate a high prevalence of both types of SIVmnd; all 10 sexually mature wild-living monkeys and 3 out of 17 wild-born juveniles tested were infected. The geographic distribution of SIVmnd seems to be distinct for the two types: SIVmnd-1 viruses were exclusively identified in mandrills from central and southern Gabon, whereas SIVmnd-2 viruses were identified in monkeys from northern and western Gabon, as well as in Cameroon. SIVmnd-2 full-length sequence analysis, together with analysis of partial sequences from SIVmnd-1 and SIVmnd-2 from wild-born or wild-living mandrills, shows that the gag and pol regions of SIVmnd-2 are closest to those of SIVrcm, isolated from red-capped mangabeys (Cercocebus torquatus), while the env gene is closest to that of SIVmnd-1. pol and env sequence analyses of SIV from a related Papionini species, the drill (Mandrillus leucophaeus), shows a closer relationship of SIVdrl to SIVmnd-2 than to SIVmnd-1. Epidemiological surveys of human immunodeficiency virus revealed a case in Cameroon of a human infected by a virus serologically related to SIVmnd, raising the possibility that mandrills represent a viral reservoir for humans similar to sooty mangabeys in Western Africa and chimpanzees in Central Africa.

We developed a simple, rapid, inexpensive, and highly sensitive and specific strategy for the detection and lineage differentiation of primate lentiviruses (PIV-ELISA). It is based on the use of two indirect ELISA methods using synthetic peptides mapping the gp41/36 region (detection component) and the V3 region (differentiation component) of four lentivirus lineages, namely SIVcpz/HIV-1 (groups M, O, N, and SIVcpz-gab), SIVmnd, SIVagm, and SIVsm/SIVmac/HIV-2. This strategy was evaluated with panels of sera originating from both humans and nonhuman primates. The human reference panel consisted of 144 HIV Western blot (WB)-positive sera in which the corresponding virus had been genotyped (HIV-1: 72 group M, 28 group O, and 6 group N; HIV-2: 21 subtype A and 10 subtype B; and 7 HIV-1+2) and 105 HIV WB-negative samples. The nonhuman primate reference panel consisted of 24 sera from monkeys infected by viruses belonging to the four lineages included in the PIV-ELISA strategy (5 chimpanzees, 5 macaques, 8 mandrills, and 6 vervets) and 42 samples from seronegative animals. Additional field evaluation panels consisted of 815 human sera from Gabon, Cameroon, and France and 537 samples from 25 nonhuman primate species. All the samples from the two reference panels were correctly detected and discriminated by PIV-ELISA. In the human field evaluation panel, the gp41/36 component correctly identified all the test samples, with 98% specificity. The V3 component discriminated 206 HIV-1 group M, 98 group O, 12 group M+O, and 128 HIV-2 sera. In the primate field evaluation panel, both gp41/36 and V3 detected and discriminated all the WB-positive samples originating from monkeys infected with SIVcpz, SIVagm-ver, SIVmnd-1, SIVmnd-2, SIVdrl, or SIVsun. These results were confirmed by genotyping in every case. Four SIV-infected red-capped mangabeys (confirmed by PCR) were correctly identified by gp41/36, but only two reacted with the V3 peptides in the absence of a specific SIVrcm V3 peptide. Addition of a V3 SIVrcm peptide discriminated all the SIVrcm-positive samples. Fourteen Papio papio samples were positive for SIVsm gp 36 and by WB, but negative by PCR, whereas three Papio cynocephalus samples were positive by gp41/36 but indeterminate by WB and negative by PCR. This combined ELISA system is thus highly sensitive and specific for antibodies directed against HIV and SIV. In addition, the V3-based serotyping results always agreed with genotyping results. This method should prove useful for studies of lentivirus prevalence and diversity in human and nonhuman primates, and may also have the potential to detect previously undescribed SIVs.

In contrast to pathogenic human immunodeficiency virus and simian immunodeficiency virus (SIV) infections, chronic SIVagm infections in African green monkeys (AGMs) are characterized by persistently low peripheral and tissue viral loads that correlate with the lack of disease observed in these animals. We report here data on the dynamics of acute SIVagm infection in AGMs that exhibit remarkable similarities with viral replication patterns observed in peripheral blood during the first 2 weeks of pathogenic SIVmac infections. Plasma viremia was evident at day 3 postinfection (p.i.) in AGMs, and rapid viral replication led by days 7 to 10 to peak viremias characterized by high levels of antigenemia (1.2 to 5 ng of p27/ml of plasma), peripheral DNA viral load (10(4) to 10(5) DNA copies/10(6) peripheral blood mononuclear cells [PBMC]), and plasma RNA viral load (2 x 10(6) to 2 x 10(8) RNA copies/ml). The lymph node (LN) RNA and DNA viral load patterns were similar to those in blood, with peaks observed between day 7 and day 14. These values in LNs (ranging from 3 x 10(5) to 3 x 10(6) RNA copies/10(6) LN cell [LNC] and 10(3) to 10(4) DNA copies/10(6) LNC) were at no time point higher than those observed in the blood. Both in LNs and in blood, rapid and significant decreases were observed in all infected animals after this peak of viral replication. Within 3 to 4 weeks p. i., antigenemia was no longer detectable and peripheral viral loads decreased to values similar to those characteristic of the chronic phase of infection (10(2) to 10(3) DNA copies/10(6) PBMC and 2 x 10(3) to 2 x 10(5) RNA copies/ml of plasma). In LNs, viral loads declined to 5 x 10(1) to 10(3) DNA copies and 10(4) to 3 x 10(5) RNA copies per 10(6) LNC at day 28 p.i. and continued to decrease until day 84 p.i. (<10 to 3 x 10(4) RNA copies/10(6) LNC). Despite extensive viremia during primary infection, neither follicular hyperplasia nor CD8(+) cell infiltration into LN germinal centers was detected. Altogether, these results indicate that the nonpathogenic outcome of SIVagm infection in its natural host is associated with a rapidly induced control of viral replication in response to SIVagm infection, rather than with a poorly replicating virus or a constitutive host genetic resistance to virus replication.

African monkeys can be naturally infected with SIV but do not progress to AIDS. Since mutations in the human CCR5 gene have been shown to influence susceptibility to HIV infection and disease progression, we have now investigated whether mutations in CCR5-coding sequences in African nonhuman primates can explain species-specific differences in susceptibility to lentiviral infection. The animals studied comprise chronically infected monkeys corresponding to four natural hosts of SIV (Cercopithecus aethiops, Cercopithecus pygerythrus, Cercopithecus sabaeus, and Cercopithecus tantalus), noninfected animals from three species that are known to be susceptible to SIV infection (Cercopithecus patas, Cercopithecus Ihoesti, and Pan troglodytes), and monkeys of six species that do not carry SIV in the wild (Cercocebus galeritus, Cercocebus aterrimus, Cercopithecus ascanius, Cercopithecus nictitans, Cercopithecus neglectus, and Cercopithecus cephus). We observed a high degree of genetic divergence among the species. The rate of accumulation of amino acid mutations was, however, not higher in SIV carriers than in other nonhuman primates. No homozygous premature stop codons, deletions, or frameshift mutations were detected. In at least two animals, one infected AGM (Cercopithecus tantalus) and one noninfected monkey (Cercocebus aterrimus), the CCR5 alleles identified encode functional proteins, as they were identical in terms of amino acid sequence to that of functional CCR5 reported in the literature. We found no other consistent differences in the genetic variability of CCR5-coding sequences between the nonhuman primates that are carriers of SIV and those that are not.

The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants.

To investigate the genetic and biologic features of HIV-1 strains circulating in Cambodia, viruses from 95 HIV-1-seropositive individuals were subtyped by heteroduplex mobility assay (HMA) and 23 were further analyzed for their biologic characteristics. Eighty-nine individuals were clearly infected by HIV-1 subtype E. The other six samples were sequenced, together with 17 HMA subtype E samples. All but one of the 23 Cambodian env sequences clustered with previously described Thai and Vietnamese subtype E sequences, bearing a GPGQ motif at the tip of the V3 loop; the last had a GPGR motif and was phylogenetically equidistant from Asian and African subtype E viruses. Nonsyncytium-inducing, CCR5-dependent viruses predominated in patients of clinical stage B even in some with a high viral load and were detected in about 50% of the patients of stage C. All syncytium-inducing strains, mostly from AIDS patients, used both CCR5 and CXCR4. The presence of syncytium-inducing viruses did not correlate with the plasma viral load. These data show that CCR5-dependent HIV-1 subtype E is currently predominant in Cambodia. The analysis of clinical and virologic markers strongly supports the idea that dynamics of the viral population during subtype E infection in Southeast Asia is similar to that of subtype B infection in Europe and the United States.

A highly divergent HIV-1 isolate, designated YBF 30, was obtained in 1995 from a 40-year-old Cameroonian woman with AIDS. Depending on the genes studied, phylogenetic analysis showed that YBF30 branched either with SIVcpz-gab or between SIVcpz-gab and HIV-1 group M. The structural genes and tat, vpr, and nef of YBF30 are approximately equidistant from those of HIV-1 group M and SIVcpz-gab. In contrast, vif and rev are closer to HIV-1 group M, and vpu is highly divergent. Using a YBF30 V3 loop peptide enzyme immunoassay, we screened 700 HIV-1-positive sera collected in Cameroon; three reacted strongly with the YBF30 peptides and one was confirmed as being related to YBF30 by genetic analysis of a pol fragment. YBF30 is as distinct from SIVcpz-gab as it is from HIV-1 group M and can thus be considered as the prototype strain of a new human immunodeficiency virus group.

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son's virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.

Natural SIVmnd and STLVmnd infections of mandrills in a colony at the Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon were investigated by genetic analysis to determine the extent of intracolony transmission. SIVmnd pol sequence analysis indicates that the six strains present in the colony belong to the SIVmnd lentivirus subgroup previously defined according to the only available prototype sequence (SIVmndGB1), which originated from the same colony. The intraanimal nucleotide diversity (1.1-3.1%) was similar in range to that reported in individuals infected by other HIV/SIVs. The interanimal diversity (0.5-4.3%) was not significantly different from that observed in each individual mandrill, indicating an epidemiological link among the SIVmnd isolates of distinct animals within the colony. Phylogenetic analysis of these isolates, together with seroepidemiological and behavior surveillance within the colony, indicates a predominant male-to-male transmission of SIVmnd that probably occurred during bouts of interanimal aggression. Moreover, our results suggest one case of vertical transmission of SIVmnd from a naturally infected founder female to one of her six offspring. The first genetic analysis of STLV isolates from mandrills is also reported here. Partial tax/rex sequences were used to evaluate the diversity between seven STLVmnd isolates and their phylogenetic relationships with other known strains of human and nonhuman primate T cell leukemia virus, types I and II (PTLV-I/II). They all belong to the PTLV-I subtype, but two genetically distinct STLVmnd groups were evidenced within the mandrill colony. The phylogenetic analyses of the STLVmnd isolates, together with seroepidemiological and behavior surveillance of the mandrills, indicate that intracolony transmissions of STLVmnd are also predominantly the result of male-to-male aggressive contacts.

An apparent species-specific relatedness of SIVagm suggests a coevolution with their natural hosts. However, the exact species or subspecies classification of African green monkeys, AGM, is uncertain because current classification schemes rely on phenotype markers, while more definitive genetic data are lacking. In this study, the CD4 protein involved in tissue type recognition was genetically cloned and sequenced from PBMC RNA from all AGM species, including Barbados green monkeys (BGM). Phylogenetic trees were constructed that also included genomic CD4 nucleotide sequences from patas, sooty mangabeys, rhesus and pig-tail macaques, chimpanzees, and humans. Chimpanzees and humans consistently clustered together. Monkeys within the Cercopithecus genus formed a separate cluster which included pata monkeys, supporting its grouping as a member of Cercopithecus. Surprisingly, sooty mangabeys were genetically more closely related to Asian macaques than to other African species, which might explain why macaques are more susceptible to infection by the SIVsm group than to infection by SIVagm or HIV-1 and why patas, on the other hand, are highly susceptible to SIVagm infection. Based on CD4 genetic data, tantalus, vervets, grivets, and sabaeus formed separate subgroups with BGM grouping closely with vervets. The branching order of the AGM species was related to that of their respective SIVagm env sequences. The study suggests a strong correlation between CD4 phylogeny and the susceptibility of the host species to infection by a specific lentivirus and supports the assumption of a coevolution of SIVagm and AGM. CD4 sequencing is suggested as a relevant method for genetic determination of primate species.

African green monkeys (AGMs) are divided into four species (Cercopithecus aethiops, C. pygerythrus, C. sabaeus, C. tantalus), each harboring a species-specific simian immunodeficiency virus (SIVagm). Little is known about the host and/or viral factors that are responsible for the apathogenicity of SIVagm infections in their natural hosts. In order to analyze the specific selective pressures exerted by the host on the virus in vivo, we compared the genetic evolution of SIVagm.tan in its natural host (C. tantalus) and in a foreign host species (Erythrocebus patas), in which we could obtain a reproducible and persistent infection by SIVagm.tan. As in AGMs, patas monkeys do not develop any disease following SIVagm infection. Our longitudinal study in env (V3-C3-V4-C4-V5) of SIVagm.tan from three AGMs and three patas monkeys revealed a high ratio of synonymous to nonsynonymous mutation frequencies (1.5-6.2). These data indicate that the selective pressures for stability exerted by AGMs and patas monkeys on SIVagm override positive selection for change reported in pathogenic HIV-1 infections. The rapid accumulation of mutations observed in AGMs and patas monkeys (0.4-7.2 x 10(-2) nucleotide substitutions per site per year) suggests a continuous replication of SIVagm viruses in vivo. We thus propose that nonpathogenic SIVagm infections are the result of a long-term selection of SIVagm variants whose dissemination can be controlled in the host, rather than being explained by a low ability of the virus to replicate in vivo.