Ape Origins of Human Malaria.

African apes harbor at least twelve Plasmodium species, some of which have been a source of human infection. It is now well established that Plasmodium falciparum emerged following the transmission of a gorilla parasite, perhaps within the last 10,000 years, while Plasmodium vivax emerged earlier from a parasite lineage that infected humans and apes in Africa before the Duffy-negative mutation eliminated the parasite from humans there. Compared to their ape relatives, both human parasites have greatly reduced genetic diversity and an excess of nonsynonymous mutations, consistent with severe genetic bottlenecks followed by rapid population expansion. A putative new Plasmodium species widespread in chimpanzees, gorillas, and bonobos places the origin of Plasmodium malariae in Africa. Here, we review what is known about the origins and evolutionary history of all human-infective Plasmodium species, the time and circumstances of their emergence, and the diversity, host specificity, and zoonotic potential of their ape counterparts.

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

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.

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d'Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally.

Chemically directed structure evolution for crystal structure prediction.

We present a new method of evolving crystal structures for crystal structure prediction. The method of chemically directed structure evolution uses chemical models to quantify the environment of atoms and vacancy sites in a crystal structure, with that information used to inform how to modify the structure to make a move on the potential energy surface. We have developed a method of chemically directed swapping, where we swap atoms in the least favourable chemical environments. This method has been implemented in the crystal structure prediction code ChemDASH (Chemically Directed Atom Swap Hopping), which explores the potential energy surface using a basin-hopping approach, and evaluates chemical environments using either the bond valence sum or electrostatic site potential. ChemDASH has a variety of methods of initialising structures, optimising structures, and swapping atoms. This gives ChemDASH the flexibility to be applied to a wide range of systems. We used ChemDASH to examine the effectiveness of the directed swapping method. Directed swapping finds the ground states of TiO2 and SrTiO3 faster than random (non-directed) swapping, but is less effective than random swapping for Y2Ti2O7.

Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness.

Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average threefold more infectious (P = 0.0001), replicated to 1.4-fold higher titers (P = 0.004), were released from infected cells 4.2-fold more efficiently (P < 0.00001), and were significantly more resistant to type I IFNs than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P < 0.00001) and 39-fold higher IFNß (P < 0.00001) half-maximal inhibitory concentrations (IC50) than did donor isolates, and their odds of replicating in CD4+ T cells at the highest IFNα2 and IFNß doses were 35-fold (P < 0.00001) and 250-fold (P < 0.00001) greater, respectively. Interestingly, pretreatment of CD4+ T cells with IFNß, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.

Origin of the HIV-1 group O epidemic in western lowland gorillas.

HIV-1, the cause of AIDS, is composed of four phylogenetic lineages, groups M, N, O, and P, each of which resulted from an independent cross-species transmission event of simian immunodeficiency viruses (SIVs) infecting African apes. Although groups M and N have been traced to geographically distinct chimpanzee communities in southern Cameroon, the reservoirs of groups O and P remain unknown. Here, we screened fecal samples from western lowland (n = 2,611), eastern lowland (n = 103), and mountain (n = 218) gorillas for gorilla SIV (SIVgor) antibodies and nucleic acids. Despite testing wild troops throughout southern Cameroon (n = 14), northern Gabon (n = 16), the Democratic Republic of Congo (n = 2), and Uganda (n = 1), SIVgor was identified at only four sites in southern Cameroon, with prevalences ranging from 0.8-22%. Amplification of partial and full-length SIVgor sequences revealed extensive genetic diversity, but all SIVgor strains were derived from a single lineage within the chimpanzee SIV (SIVcpz) radiation. Two fully sequenced gorilla viruses from southwestern Cameroon were very closely related to, and likely represent the source population of, HIV-1 group P. Most of the genome of a third SIVgor strain, from central Cameroon, was very closely related to HIV-1 group O, again pointing to gorillas as the immediate source. Functional analyses identified the cytidine deaminase APOBEC3G as a barrier for chimpanzee-to-gorilla, but not gorilla-to-human, virus transmission. These data indicate that HIV-1 group O, which spreads epidemically in west central Africa and is estimated to have infected around 100,000 people, originated by cross-species transmission from western lowland gorillas.

Effective treatment of SIVcpz-induced immunodeficiency in a captive western chimpanzee.

BACKGROUND: Simian immunodeficiency virus of chimpanzees (SIVcpz), the progenitor of human immunodeficiency virus type 1 (HIV-1), is associated with increased mortality and AIDS-like immunopathology in wild-living chimpanzees (Pan troglodytes). Surprisingly, however, similar findings have not been reported for chimpanzees experimentally infected with SIVcpz in captivity, raising questions about the intrinsic pathogenicity of this lentivirus. FINDINGS: Here, we report progressive immunodeficiency and clinical disease in a captive western chimpanzee (P. t. verus) infected twenty years ago by intrarectal inoculation with an SIVcpz strain (ANT) from a wild-caught eastern chimpanzee (P. t. schweinfurthii). With sustained plasma viral loads of 105 to 106 RNA copies/ml for the past 15 years, this chimpanzee developed CD4+ T cell depletion (220 cells/µl), thrombocytopenia (90,000 platelets/µl), and persistent soft tissue infections refractory to antibacterial therapy. Combination antiretroviral therapy consisting of emtricitabine (FTC), tenofovir disoproxil fumarate (TDF), and dolutegravir (DTG) decreased plasma viremia to undetectable levels (<200 copies/ml), improved CD4+ T cell counts (509 cell/µl), and resulted in the rapid resolution of all soft tissue infections. However, initial lack of adherence and/or differences in pharmacokinetics led to low plasma drug concentrations, which resulted in transient rebound viremia and the emergence of FTC resistance mutations (M184V/I) identical to those observed in HIV-1 infected humans. CONCLUSIONS: These data demonstrate that SIVcpz can cause immunodeficiency and other hallmarks of AIDS in captive chimpanzees, including P. t. verus apes that are not naturally infected with this virus. Moreover, SIVcpz-associated immunodeficiency can be effectively treated with antiretroviral therapy, although sufficiently high plasma concentrations must be maintained to prevent the emergence of drug resistance. These findings extend a growing body of evidence documenting the immunopathogenicity of SIVcpz and suggest that experimentally infected chimpanzees may benefit from clinical monitoring and therapeutic intervention.

Morphological and Genetic Analyses of the Invasive Forest Pathogen Phytophthora austrocedri Reveal that Two Clonal Lineages Colonized Britain and Argentina from a Common Ancestral Population.

Phytophthora austrocedri is causing widespread mortality of Austrocedrus chilensis in Argentina and Juniperus communis in Britain. The pathogen has also been isolated from J. horizontalis in Germany. Isolates from Britain, Argentina, and Germany are homothallic, with no clear differences in the dimensions of sporangia, oogonia, or oospores. Argentinian and German isolates grew faster than British isolates across a range of media and had a higher temperature tolerance, although most isolates, regardless of origin, grew best at 15°C and all isolates were killed at 25°C. Argentinian and British isolates caused lesions when inoculated onto both A. chilensis and J. communis; however, the Argentinian isolate caused longer lesions on A. chilensis than on J. communis and vice versa for the British isolate. Genetic analyses of nuclear and mitochondrial loci showed that all British isolates are identical. Argentinian isolates and the German isolate are also identical but differ from the British isolates. Single-nucleotide polymorphisms are shared between the British and Argentinian isolates. We concluded that British isolates and Argentinian isolates conform to two distinct clonal lineages of P. austrocedri founded from the same as-yet-unidentified source population. These lineages should be recognized and treated as separate risks by international plant health legislation.

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.

Plasmodium falciparum-like parasites infecting wild apes in southern Cameroon do not represent a recurrent source of human malaria.

Wild-living chimpanzees and gorillas harbor a multitude of Plasmodium species, including six of the subgenus Laverania, one of which served as the progenitor of Plasmodium falciparum. Despite the magnitude of this reservoir, it is unknown whether apes represent a source of human infections. Here, we used Plasmodium species-specific PCR, single-genome amplification, and 454 sequencing to screen humans from remote areas of southern Cameroon for ape Laverania infections. Among 1,402 blood samples, we found 1,000 to be Plasmodium mitochondrial DNA (mtDNA) positive, all of which contained human parasites as determined by sequencing and/or restriction enzyme digestion. To exclude low-abundance infections, we subjected 514 of these samples to 454 sequencing, targeting a region of the mtDNA genome that distinguishes ape from human Laverania species. Using algorithms specifically developed to differentiate rare Plasmodium variants from 454-sequencing error, we identified single and mixed-species infections with P. falciparum, Plasmodium malariae, and/or Plasmodium ovale. However, none of the human samples contained ape Laverania parasites, including the gorilla precursor of P. falciparum. To characterize further the diversity of P. falciparum in Cameroon, we used single-genome amplification to amplify 3.4-kb mtDNA fragments from 229 infected humans. Phylogenetic analysis identified 62 new variants, all of which clustered with extant P. falciparum, providing further evidence that P. falciparum emerged following a single gorilla-to-human transmission. Thus, unlike Plasmodium knowlesi-infected macaques in southeast Asia, African apes harboring Laverania parasites do not seem to serve as a recurrent source of human malaria, a finding of import to ongoing control and eradication measures.

RNA-mediated degradation of microRNAs: A widespread viral strategy?

Regulation of small RNAs by other non-coding RNAs is a ubiquitous feature of gene regulatory systems that can be exploited by viruses. Examples of this have been described in 3 different herpesviruses, where viral non-coding RNAs bind to highly abundant cellular (miRNAs), mediating their degradation: miR-27 is targeted by both murine cytomegalovirus and herpesvirus saimiri, while the miR-17 family is targeted by human cytomegalovirus. We review what is known about RNA-mediated regulation of miRNA stability and propose 3 potential roles that viral non-coding RNAs might assume to initiate the destruction of a miRNA, acting as "recruiters," "localizers" or "exposers." Whereas the miRNAs (miR-17 and miR-27) appear to be ancient and pre-date the common ancestor of all mammalian herpesviruses, comparative analyses of herpesvirus genomes indicate that the 3 known viral regulators of miRNA each evolved independently, and much more recently. Noting that the anti-viral activity of miRNAs might be countered by a variety of mechanisms, we propose that (i) there has been continual turnover of these mechanisms during herpesvirus evolution, and (ii) there may be many other, as yet undescribed, anti-miRNA activities encoded by other herpesviruses and indeed by viruses from other families.

Increased mortality and AIDS-like immunopathology in wild chimpanzees infected with SIVcpz.

African primates are naturally infected with over 40 different simian immunodeficiency viruses (SIVs), two of which have crossed the species barrier and generated human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Unlike the human viruses, however, SIVs do not generally cause acquired immunodeficiency syndrome (AIDS) in their natural hosts. Here we show that SIVcpz, the immediate precursor of HIV-1, is pathogenic in free-ranging chimpanzees. By following 94 members of two habituated chimpanzee communities in Gombe National Park, Tanzania, for over 9 years, we found a 10- to 16-fold higher age-corrected death hazard for SIVcpz-infected (n = 17) compared to uninfected (n = 77) chimpanzees. We also found that SIVcpz-infected females were less likely to give birth and had a higher infant mortality rate than uninfected females. Immunohistochemistry and in situ hybridization of post-mortem spleen and lymph node samples from three infected and two uninfected chimpanzees revealed significant CD4(+) T-cell depletion in all infected individuals, with evidence of high viral replication and extensive follicular dendritic cell virus trapping in one of them. One female, who died within 3 years of acquiring SIVcpz, had histopathological findings consistent with end-stage AIDS. These results indicate that SIVcpz, like HIV-1, is associated with progressive CD4(+) T-cell loss, lymphatic tissue destruction and premature death. These findings challenge the prevailing view that all natural SIV infections are non-pathogenic and suggest that SIVcpz has a substantial negative impact on the health, reproduction and lifespan of chimpanzees in the wild.

The two parasite species formerly known as Plasmodium ovale.

Plasmodium ovale was the last of the exclusively human malaria parasites to be described, in 1922, and has remained the least well studied. Beginning in 1995, two divergent forms of the parasite, later termed 'classic' and 'variant', were described. By 2010, it was realised that these forms are two closely related, but genetically distinct and non-recombining species; they were given the names Plasmodium ovale curtisi and Plasmodium ovale wallikeri. Since then, substantial additional data have confirmed that the two parasites are indeed separate species, but the trinomial nomenclature has often led to confusion about their status, with many authors describing them as subspecies. We hereby formally name them Plasmodium ovalecurtisi and Plasmodium ovalewallikeri.

Origin of the human malaria parasite Plasmodium vivax.

The geographic origin of Plasmodium vivax, a leading cause of human malaria, has been the subject of much speculation. Here we review the evolutionary history of P. vivax and P. vivax-like parasites in humans and non-human primates on three continents, providing overwhelming evidence for an African origin. This conclusion is consistent with recent reports showing that Duffy-negative humans in Africa are, in fact, susceptible to P. vivax, with parasites invading Duffy-antigen-expressing erythroid precursors. Thus, the African origin of P. vivax not only explains the distribution of the Duffy-negative genotype but also provides new insight into the history and status of P. vivax malaria in Africa and efforts geared toward its eradication.

Eastern chimpanzees, but not bonobos, represent a simian immunodeficiency virus reservoir.

Chimpanzees in west central Africa (Pan troglodytes troglodytes) are endemically infected with simian immunodeficiency viruses (SIVcpzPtt) that have crossed the species barrier to humans and gorillas on at least five occasions, generating pandemic and nonpandemic forms of human immunodeficiency virus type 1 (HIV-1) as well as gorilla SIV (SIVgor). Chimpanzees in east Africa (Pan troglodytes schweinfurthii) are also infected with SIVcpz; however, their viruses (SIVcpzPts) have never been found in humans. To examine whether this is due to a paucity of natural infections, we used noninvasive methods to screen wild-living eastern chimpanzees in the Democratic Republic of the Congo (DRC), Uganda, and Rwanda. We also screened bonobos (Pan paniscus) in the DRC, a species not previously tested for SIV in the wild. Fecal samples (n = 3,108) were collected at 50 field sites, tested for species and subspecies origin, and screened for SIVcpz antibodies and nucleic acids. Of 2,565 samples from eastern chimpanzees, 323 were antibody positive and 92 contained viral RNA. The antibody-positive samples represented 76 individuals from 19 field sites, all sampled north of the Congo River in an area spanning 250,000 km(2). In this region, SIVcpzPts was common and widespread, with seven field sites exhibiting infection rates of 30% or greater. The overall prevalence of SIVcpzPts infection was 13.4% (95% confidence interval, 10.7% to 16.5%). In contrast, none of the 543 bonobo samples from six sites was antibody positive. All newly identified SIVcpzPts strains clustered in strict accordance to their subspecies origin; however, they exhibited considerable genetic diversity, especially in protein domains known to be under strong host selection pressure. Thus, the absence of SIVcpzPts zoonoses cannot be explained by an insufficient primate reservoir. Instead, greater adaptive hurdles may have prevented the successful colonization of humans by P. t. schweinfurthii viruses.

Unusually Divergent Ubiquitin Genes and Proteins in Plasmodium Species.

Ubiquitin is an extraordinarily highly conserved 76 amino acid protein encoded by three different types of gene, where the primary translation products are fusions either of ubiquitin with one of two ribosomal proteins (RPs) or of multiple ubiquitin monomers from head to tail. Here, we investigate the evolution of ubiquitin genes in mammalian malaria parasites (Plasmodium species). The ubiquitin encoded by the RPS27a fusion gene is highly divergent, as previously found in a variety of protists. However, we also find that two other forms of divergent ubiquitin sequence, each previously thought to be extremely rare, have arisen recently during the divergence of Plasmodium subgenera. On two occasions, in two distinct lineages, the ubiquitin encoded by the RPL40 fusion gene has rapidly diverged. In addition, in one of these lineages, the polyubiquitin genes have undergone a single codon insertion, previously considered a unique feature of Rhizaria. There has been disagreement whether the multiple ubiquitin coding repeats within a genome exhibit concerted evolution or undergo a birth-and-death process; the Plasmodium ubiquitin genes show clear signs of concerted evolution, including the spread of this codon insertion to multiple repeats within the polyubiquitin gene.

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.