Widespread extinctions of co-diversified primate gut bacterial symbionts from humans.

Humans and other primates harbour complex gut bacterial communities that influence health and disease, but the evolutionary histories of these symbioses remain unclear. This is partly due to limited information about the microbiota of ancestral primates. Here, using phylogenetic analyses of metagenome-assembled genomes (MAGs), we show that hundreds of gut bacterial clades diversified in parallel (that is, co-diversified) with primate species over millions of years, but that humans have experienced widespread losses of these ancestral symbionts. Analyses of 9,460 human and non-human primate MAGs, including newly generated MAGs from chimpanzees and bonobos, revealed significant co-diversification within ten gut bacterial phyla, including Firmicutes, Actinobacteriota and Bacteroidota. Strikingly, ~44% of the co-diversifying clades detected in African apes were absent from available metagenomic data from humans and ~54% were absent from industrialized human populations. In contrast, only ~3% of non-co-diversifying clades detected in African apes were absent from humans. Co-diversifying clades present in both humans and chimpanzees displayed consistent genomic signatures of natural selection between the two host species but differed in functional content from co-diversifying clades lost from humans, consistent with selection against certain functions. This study discovers host-species-specific bacterial symbionts that predate hominid diversification, many of which have undergone accelerated extinctions from human populations.

Malaria-driven adaptation of MHC class I in wild bonobo populations.

The malaria parasite Plasmodium falciparum causes substantial human mortality, primarily in equatorial Africa. Enriched in affected African populations, the B*53 variant of HLA-B, a cell surface protein that presents peptide antigens to cytotoxic lymphocytes, confers protection against severe malaria. Gorilla, chimpanzee, and bonobo are humans' closest living relatives. These African apes have HLA-B orthologs and are infected by parasites in the same subgenus (Laverania) as P. falciparum, but the consequences of these infections are unclear. Laverania parasites infect bonobos (Pan paniscus) at only one (TL2) of many sites sampled across their range. TL2 spans the Lomami River and has genetically divergent subpopulations of bonobos on each side. Papa-B, the bonobo ortholog of HLA-B, includes variants having a B*53-like (B07) peptide-binding supertype profile. Here we show that B07 Papa-B occur at high frequency in TL2 bonobos and that malaria appears to have independently selected for different B07 alleles in the two subpopulations.

The ecology and epidemiology of malaria parasitism in wild chimpanzee reservoirs.

Chimpanzees (Pan troglodytes) harbor rich assemblages of malaria parasites, including three species closely related to P. falciparum (sub-genus Laverania), the most malignant human malaria parasite. Here, we characterize the ecology and epidemiology of malaria infection in wild chimpanzee reservoirs. We used molecular assays to screen chimpanzee fecal samples, collected longitudinally and cross-sectionally from wild populations, for malaria parasite mitochondrial DNA. We found that chimpanzee malaria parasitism has an early age of onset and varies seasonally in prevalence. A subset of samples revealed Hepatocystis mitochondrial DNA, with phylogenetic analyses suggesting that Hepatocystis appears to cross species barriers more easily than Laverania. Longitudinal and cross-sectional sampling independently support the hypothesis that mean ambient temperature drives spatiotemporal variation in chimpanzee Laverania infection. Infection probability peaked at ~24.5 °C, consistent with the empirical transmission optimum of P. falciparum in humans. Forest cover was also positively correlated with spatial variation in Laverania prevalence, consistent with the observation that forest-dwelling Anophelines are the primary vectors. Extrapolating these relationships across equatorial Africa, we map spatiotemporal variation in the suitability of chimpanzee habitat for Laverania transmission, offering a hypothetical baseline indicator of human exposure risk.

CD4 receptor diversity in chimpanzees protects against SIV infection.

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.

Wild bonobos host geographically restricted malaria parasites including a putative new Laverania species.

Malaria parasites, though widespread among wild chimpanzees and gorillas, have not been detected in bonobos. Here, we show that wild-living bonobos are endemically Plasmodium infected in the eastern-most part of their range. Testing 1556 faecal samples from 11 field sites, we identify high prevalence Laverania infections in the Tshuapa-Lomami-Lualaba (TL2) area, but not at other locations across the Congo. TL2 bonobos harbour P. gaboni, formerly only found in chimpanzees, as well as a potential new species, Plasmodium lomamiensis sp. nov. Rare co-infections with non-Laverania parasites were also observed. Phylogenetic relationships among Laverania species are consistent with co-divergence with their gorilla, chimpanzee and bonobo hosts, suggesting a timescale for their evolution. The absence of Plasmodium from most field sites could not be explained by parasite seasonality, nor by bonobo population structure, diet or gut microbiota. Thus, the geographic restriction of bonobo Plasmodium reflects still unidentified factors that likely influence parasite transmission.

Bonobos Maintain Immune System Diversity with Three Functional Types of MHC-B.

Fast-evolving MHC class I polymorphism serves to diversify NK cell and CD8 T cell responses in individuals, families, and populations. Because only chimpanzee and bonobo have strict orthologs of all HLA class I, their study gives unique perspectives on the human condition. We defined polymorphism of Papa-B, the bonobo ortholog of HLA-B, for six wild bonobo populations. Sequences for Papa-B exon 2 and 3 were determined from the genomic DNA in 255 fecal samples, minimally representing 110 individuals. Twenty-two Papa-B alleles were defined, each encoding a different Papa-B protein. No Papa-B is identical to any chimpanzee Patr-B, human HLA-B, or gorilla Gogo-B. Phylogenetic analysis identified a clade of MHC-B, defined by residues 45-74 of the α1 domain, which is broadly conserved among bonobo, chimpanzee, and gorilla. Bonobo populations have 3-14 Papa-B allotypes. Three Papa-B are in all populations, and they are each of a different functional type: allotypes having the Bw4 epitope recognized by killer cell Ig-like receptors of NK cells, allotypes having the C1 epitope also recognized by killer cell Ig-like receptors, and allotypes having neither epitope. For population Malebo, these three Papa-B are the only Papa-B allotypes. Although small in number, their sequence divergence is such that the nucleotide diversity (mean proportional distance) of Papa-B in Malebo is greater than in the other populations and is also greater than expected for random combinations of three Papa-B Overall, Papa-B has substantially less diversity than Patr-B in chimpanzee subspecies and HLA-B in indigenous human populations, consistent with bonobo having experienced narrower population bottlenecks.

Genetic diversity of STLV-2 and interspecies transmission of STLV-3 in wild-living bonobos.

There are currently four known primate T-cell lymphotropic virus groups (PTLV1-4), each of which comprises closely related simian (STLV) and human (HTLV) viruses. For PTLV-1 and PTLV-3, simian and human viruses are interspersed, suggesting multiple cross-species transmission events; however, for PTLV-2 this is not so clear because HTLV-2 and STLV-2 strains from captive bonobos (Pan paniscus) form two distinct clades. To determine to what extent bonobos are naturally infected with STLV, we screened fecal samples (n = 633) from wild-living bonobos (n = 312) at six different sites in the Democratic Republic of Congo (DRC) for the presence of STLV nucleic acids. STLV infection was detected in 8 of 312 bonobos at four of six field sites, suggesting an overall prevalence of 2.6% (ranging from 0 to 8%). Six samples contained STLV-2, while the two others contained STLV-3, as determined by phylogenetic analysis of partial tax and Long Terminal Repeats (LTR) sequences. The new STLV-2 sequences were highly diverse, but grouped with previously identified STLV-2 strains as a sister clade to HTLV-2. In contrast, the new STLV-3 sequences did not cluster together, but were more closely related to STLVs from sympatric monkey species. These results show for the first time that fecal samples can be used to detect STLV infection in apes. These results also show that wild-living bonobos are endemically infected with STLV-2, but have acquired STLV-3 on at least two occasions most likely by cross-species transmission from monkey species on which they prey. Future studies of bonobos and other non-human primate species in Central Africa are needed to identify the simian precursor of HTLV-2 in humans.

Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-Living Chimpanzees and Gorillas.

Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum.

African origin of the malaria parasite Plasmodium vivax.

Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa.

Evolutionary relationships of wild hominids recapitulated by gut microbial communities.

Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.

Origin of the human malaria parasite Plasmodium falciparum in gorillas.

Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here we develop a single-genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in faecal samples from wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed and almost always made up of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas comprised parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla origin and not of chimpanzee, bonobo or ancient human origin.

Novel circular DNA viruses in stool samples of wild-living chimpanzees.

Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem-loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (approximately 1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.

Molecular epidemiology of simian immunodeficiency virus infection in wild-living gorillas.

Chimpanzees and gorillas are the only nonhuman primates known to harbor viruses closely related to HIV-1. Phylogenetic analyses showed that gorillas acquired the simian immunodeficiency virus SIVgor from chimpanzees, and viruses from the SIVcpz/SIVgor lineage have been transmitted to humans on at least four occasions, leading to HIV-1 groups M, N, O, and P. To determine the geographic distribution, prevalence, and species association of SIVgor, we conducted a comprehensive molecular epidemiological survey of wild gorillas in Central Africa. Gorilla fecal samples were collected in the range of western lowland gorillas (n = 2,367) and eastern Grauer gorillas (n = 183) and tested for SIVgor antibodies and nucleic acids. SIVgor antibody-positive samples were identified at 2 sites in Cameroon, with no evidence of infection at 19 other sites, including 3 in the range of the Eastern gorillas. In Cameroon, based on DNA and microsatellite analyses of a subset of samples, we estimated the prevalence of SIVgor to be 1.6% (range, 0% to 4.6%), which is significantly lower than the prevalence of SIVcpzPtt in chimpanzees (5.9%; range, 0% to 32%). All newly identified SIVgor strains formed a monophyletic lineage within the SIVcpz radiation, closely related to HIV-1 groups O and P, and clustered according to their field site of origin. At one site, there was evidence for intergroup transmission and a high intragroup prevalence. These isolated hot spots of SIVgor-infected gorilla communities could serve as a source for human infection. The overall low prevalence and sporadic distribution of SIVgor could suggest a decline of SIVgor in wild populations, but it cannot be excluded that SIVgor is still more prevalent in other parts of the geographical range of gorillas.

Multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces.

Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named "Cyclovirus" whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.

Molecular ecology and natural history of simian foamy virus infection in wild-living chimpanzees.

Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples (n = 724) were collected at 25 field sites throughout equatorial Africa and tested for SFVcpz-specific antibodies (n = 706) or viral nucleic acids (n = 392). SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag (616 bp), pol-RT (717 bp), and pol-IN (425 bp) sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies.

Origin of AIDS: contaminated polio vaccine theory refuted.

Despite strong evidence to the contrary, speculation continues that the AIDS virus, human immunodeficiency virus type 1 (HIV-1), may have crossed into humans as a result of contamination of the oral polio vaccine (OPV). This 'OPV/AIDS theory' claims that chimpanzees from the vicinity of Stanleyville--now Kisangani in the Democratic Republic of Congo--were the source of a simian immunodeficiency virus (SIVcpz) that was transmitted to humans when chimpanzee tissues were allegedly used in the preparation of OPV. Here we show that SIVcpz is indeed endemic in wild chimpanzees of this region but that the circulating virus is phylogenetically distinct from all strains of HIV-1, providing direct evidence that these chimpanzees were not the source of the human AIDS pandemic.