ANRS 12325

SIVs at the interface between humans and non-human primates and evaluation of the impact of SIVs on health and survival of wild ape populations infected with the ancestors of HIV-1

Responsables scientifiques 

Martine Peeters (UMI233/INSERM U1175/ UM, Montpellier, France)

Eitel Mpoudi Ngole (IMPM/CREMER/PRESICA, Yaoundé, Cameroun)

Steve Ahuka-Mundeke (INRB, Kinshasa, RDC)

Fabian Leendertz (RKI, Berlin, Allemagne)

 

CONTEXTE ET OBJECTIFS

The main objective of the present application is to study SIVs at the interface between humans and non-human primates and evaluation of the impact of SIVs on health and survival of wild ape populations infected with the ancestors of HIV-1. The specific objectives are (1) determine the prevalence and genetic diversity in gorillas, chimpanzees and bonobos to yield insight into the origin of all HIV-1 groups, to understand circumstances by which apes and humans initially acquired SIV or HIV-1, to compare host specific mechanisms of virus adaptation and pathogenicity, (2) determine the prevalence, geographic distribution of SIV in primates which are frequently hunted in order to asses which pathogens may be a risk for humans, (3) document whether other SIVs crossed the species barrier from primates to humans, (4) document to what extent SIV infection has a negative impact on the survival of  wild chimpanzee and gorilla populations

 

Durée (en nombre de mois): 
36

1. Determine the prevalence and genetic diversity in gorillas, chimpanzees and bonobos to yield insight into the origin of all HIV-1 groups, to understand circumstances by which apes and humans initially acquired SIV or HIV-1, to compare host specific mechanisms of virus adaptation and pathogenicity,

Origin of the HIV-1 group O epidemic and HIV-1 group P in western lowland gorillas. We screened fecal samples from western lowland (n=2,611), eastern lowland (n=121) and mountain gorillas (n=200) for SIVgor specific antibodies and nucleic acids. Despite testing wild troops at sites throughout southern Cameroon (n=14), northern Gabon (n=16), the Democratic Republic of the Congo (n=2) and Uganda (n=1), SIVgor was identified at only four sites in southern Cameroon with prevalence ranging from 0.8% to 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 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. Another SIVgor strain, from central Cameroon, was very closely related to HIV-1 group O across most of its genome.  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 not from chimpanzees, but from western lowland gorillas.

High Rate of Simian Immunodeficiency Virus (SIV) Infections in wild Living Chimpanzees in Northeastern Gabon. Although the first two SIVcpz were identified in wild-born chimpanzees held in captivity in Gabon at the end of 1980s, no study has been conducted so far in wild chimpanzees in Gabon. Here we analyzed 1,458 faecal samples collected in 16 different locations across the country, and we conducted follow-up missions in two of them. We found 380 SIV antibody + samples in 6 different locations in the north and northeast, with estimated SIV prevalence of 39.45%. We amplified pol and gp41 fragments and obtained 57 new SIVcpzPtt strains from three sites. Except one, all strains clustered together within a specific phylogeographic clade. These SIV+ samples have been collected nearby villages. Humans continue to encroach in ape’s territories, therefore the emergence of a new HIV in this area has to be considered.

HIV-1 group O infection in Cameroon from 2006 to 2013: prevalence, genetic diversity, evolution. After the zoonotic introduction of HIV-1 group O by gorillas into the human population, we investigated the evolution of this viral group from 2006 to 2013 in Cameroon. HIV-1 group O was found at around 0.6% indicating that the frequency of this virus in Cameroon has remained stable over the last decades. However, we found an extensive high genetic diversity within this HIV-1 group, that resulted from previous steady increase on the effective number of HIV-1 group O infections through time, and the current distribution of the circulating viral strains still does not allow classification as subtypes.

Absence of SIV infection in wild chimpanzees from West Africa (P.t.verus) and Nigeria (P.t.elliotti)

In collaboration with the group of Fabian Leendertz, 500 fecal samples were tested from 350 wild P.t.verus collected in field sites West Africa (Guinea, Senegal, Liberia, Sierra-Leone) and 150 P.t.elliotti collected at diffenrt field sites in Nigeria and Cameroon (above the Sanaga River). All smaples were screened for HV cross-reactive antibodies with INNO-LIA, but all were negative. This is the first time that a large sample set from these chimpanzee subspecies have been tested on samples collected in their natural habitat. The results confirm most likely absence of SIV infection. 

2. Determine the prevalence, geographic distribution of SIV in primates which are frequently hunted in order to asses which pathogens may be a risk for humans,

Molecular characterization of a new mosaic Simian Immunodeficiency Virus in a naturally infected tantalus monkey (Chlorocebus tantalus) from Cameroon. African green monkeys (AGMs) represent the most widely distributed nonhuman primates species in Africa. To date, only limited information is available on molecular characteristics of SIVagm infecting C. tantalus in central Africa. Here, we characterized the full-length genome of a virus infecting a naturally infected captive C. tantalus from Cameroon by amplifying and sequencing sub-genomic PCR fragments. The new SIVagmTAN-CM545 showed a mosaic structure with alternating fragments of SIVagmSAB and SIVagmTAN lineages, suggesting that the evolution of SIVagm is more complex than previously thought.

New SIV and STLV lineages NHP bushmeat from DRC. The DRC is the epicenter of the HIV-1 epidemic, but little is known about the prevalence of simian retroviruses and human exposure in DRC, where a large number of endemic monkeys and a unique ape species are present. In a previous study of around 300 DBS samples; we showed high SIV and STLV prevalences and identified new viral lineages. Here we studied an aditional 331 samples, derived from NHP bushmeat, were collected as dried blood spots (DBS, n=283) or as tissue samples (n=36) at remote forest sites in northern and eastern DRC (Mbandaka in the Equateur Province, and Goma and Walikele in Kivu Provine). Overall, 5% and 15.4% of NHP bushmeat was infected with SIV and STLV respectively. A new SIV lineage was identified in Allan’s swamp monkeys (Allenopithecus nigroviridis). Three new STLV-1 subtypes were identified in Allan’s swamp monkeys (Allenopithecus nigroviridis), blue monkeys (Cercopithecus mitis), red tailed guenons (Cercopithecus ascanius schmidti) and agile mangabeys (Cercocebus agilis). SIV and STLV prevalences varied according to species and geographic region. Overall, today 700 DBS samples were collected among NHP bushmeat in DRC, representing 13 different species at 6 different locations across the country. Overall, SIV/HIV cross-reactive antibodies were present in 20% of the samples. PCR and sequence analysis identified new SIV lineages, SIVtrc in P.tholloni, SIVasc in C.ascanius, SIVwol in C.wolfi  and SIVofm in A. nigroviridis. Moreover, we detected a high SIV prevalence in the most frequently hunted species. Full-length genome sequencing is completed for SIVasc and identified a new SIV lineage, and illustrate the complexity of SIV evolution. Characterization of other new SIVs is ongoing.Our study illustrates clearly, even on a small sample size from a limited number of geograhic areas, that our knowledge on the genetic diversity and geographic distribution of simian retroviruses is still limited and that humans continue to be exposed to relative high proportions on infected NHP bushmeat. 

 

3. Document whether other SIVs crossed the species barrier from primates to humans,

Ongoing cross-species transmission of simian retroviruses and high HIV prevalence in rural areas. More than 3000 blood samples were obtained from adults in 26 villages in southern Cameroon. In these rural forest areas, the overall HIV prevalence ranged between 6% and 9%, and was at least two times higher in women as compared to men, reaching 20% in women aged between 25 and 34 years. Genetic characterization of 188 HIV-1 strains showed 13 transmission clusters (20% of HIV-1 strains) including 2 to 10 strains in which strains derived from women predominated, with one cluster of 10 strains exclusively from women, residing in 4 different villages on a major road for logging transport. New HTLV-1 subtype F and HTLV-3 B variants related to STLVs from local NHPs were identified and SFV infection with a chimpanzee virus, was confirmed by PCR in a 65 years old male reporting hunting. We also screened more than 1000 DBS samples from persons living in rural villages in the equateur province from DRC, and identified SFV and HTLV infections. No SIV infections were identified, but our study clearly shows contemporary transmission of simian retroviruses in rural forest areas in Central Africa. The high HIV prevalence and high rates of transmission clusters in these rural areas indicate that, if a new SIV crosses the species barrier, conditions for rapid spread are present.

4. Document to what extent SIV infection has a negative impact on the survival of wild chimpanzee and gorilla populations,

Since 2007 our team collects sequential samples of gorillas groups in the national reserve of Campo Ma’n in south-west Cameroon were we identified more than 50 SIVgor positive gorillas and were we were able to reconstitute different social groups of gorillas, with parental relationships and known home ranges over time. Follow-up samples from SIV positive and negative samples are available, one invidual has been found back at a 10 years interval. Because direct monitoring of immune deficiency by measuring CD4 cell counts is not possible in faecal samples, we study other markers that are associated with immunodeficiency

Stability of the gorilla microbiome despite simian immunodeficiency virus infection. Because those SIV infections that are pathogenic have been shown to induce changes in the host’s gut microbiome, monitoring the microbiota present in fecal samples can provide a noninvasive means for studying the effects of SIV infection on the health of wild-living primates. In contrast to the microbiomes of HIV-1 infected humans and SIVcpz-infected chimpanzees, SIVgor-infected gorilla microbiomes exhibit neither rises in the frequencies of opportunistic pathogens nor elevated rates of microbial turnover within individual hosts. Regardless of SIV infection status, gorilla microbiomes assort into enterotypes, one of which is compositionally analogous to those identified in humans and chimpanzees and another one that appears specialized for a leaf-based diet and is enriched in environmentally derived bacterial genera.

 

Gorilla viriome and SIV infection.

The expansion of the enteric virome associated with disease progression in an AIDS-like context in rhesus macaques has been described, an evidence not seen in the non-pathogenic SIV infection of African green monkey. The aim of this study was to identify and compare enteric viromes of SIV-infected and uninfected gorillas, and to assess their relationship with SIV pathogenesis.We analyzed fecal samples of 22 wild gorillas from Cameroon, 11 SIV-infected and 11 uninfected individuals from the same area. NGS was carried out in a HiSeq 2500 Illumina platform and analyses were conducted with an in-house pipeline using the programs FastQC, Sickle-Master, BlastX - Viral Database from GenBank/NCBI, MEGAN5 and RStudio. The viral families Bromoviridae, Myoviridae, Podoviridae, Rhabdoviridae and Tymoviridae were statistically (p<0.01) more abundant in the uninfected group, whereas Alloherpesviridae, Herpesviridae, Reoviridae and Siphoviridae families were more abundant (p<0.01) in the SIV-infected group. Also, two distinct clusters were recognizable when a 1,000 cp/mL cutoff of SIVgor viral load in faeces was used to assess within-group diversity. The 6 samples with higher SIVgor viral load showed Mimiviridae, Myoviridae, Parvoviridae, Phycodnaviridae, Podoviridae, Reoviridae and Retroviridae statistically (p≤0.05) more abundant families and the 5 samples with smaller viral load showed Adenoviridae, Alloherpesviridae, Inoviridae, Siphoviridae and Unclassified Phages statistically (p<0.05) more abundant taxa. Finally, we are able to detect adenovirus-assigned reads (virus associated with intestinal disease in rhesus monkeys with advanced AIDS) only in the SIV-infected samples that belong to the smaller viral load group with known infection status for at least 3 years. The enteric virome dynamics in gorillas could be potentially associated with the SIVgor status. Virome stability studies clearly provide better markers of pathogenic infection progression than bacteriomes. Further studies are still needed to better understand the influence of SIV pathogenesis on infected gorilla populations in the wild, and to associate deeper taxa (virus genera and species) to SIV status in these animals.

Transmission of SIVgor and SIV evolution over time. SIV is a virus which has a high rate of mutation and turn-over, therefore we can trace its evolution in infected individuals and epidemiologic relateness between viral strains can be studied by phylogenetic analysis (PhyML). Phylogenetic analysis of partial env and pol sequences revealed cocirculation of at least 4 SIVgor variants. Closely related and divergent strains co-circulate among gorillas from the same social group indicating SIVgor transmissions within and between groups. Vertical transmission, but horizontal transmission, by sexual or aggressive behavior, was also suspected. Intra-host molecular evolution in one gorilla over a 5-year period showed viral adaptation, characteristic of escape mutants, i.e. V1V2 loop elongation and increased number of N-glycosylation sites, studies on follow-up samples from other gorilla groups are still ongoing. Tracing the genetic variability of the virus over time will provide information on the evolution of the infection in its natural host and to study whether viral evolution patterns associated with disease in human HIV-1 infections occur also in SIVgor infecions in gorillas. This information will help us to understand to what extent this virus is affecting its host.

Perspectives

We will continue to collect samples from chimpanzees, in areas close to Kinshasa, to validate the actual hypothesis on arrival of HIV-1 group M in Kinshasa by river from southeastern Cameroon, and from bonobos in DRC, to confirm absence in this ape species, but relatively few samples have been tested today from this species.  We will continue the characterization of the new SIV lineages in NHP from DRC. We will screen additional human samples living in rural forest areas from Cameroon, DRC, and Ivory Coast for potential cross-species transmission with simian retroviruses. Finally, follow up of SIVgor infected gorilla groups will continue in the field and additional tests will be done to examine whether SIVgor has an impact on health or survival of gorillas.

 

Added Value

We have now identified the source species as well as geographic origin of all four groups of HIV-1 (M, N O and P). Both chimpanzees and gorillas represent primate lentiviral reservoirs that are capable of spawning major HIV-1 epidemics. We showed ongoing exposure to a wide diversity of simian retroviruses. SIVgor infected gorillas have apparently different viriomes compared to non-infected individuals from the same area. The samples collected from this project continue also to contribute to the identification of other important pathogens, ex. Plasmodium.

Publications n = 20

Ahuka-Mundeke S, Ayouba A, Mbala-Kingebeni P, Foncelle C, Mubonga M, Ndimbo-Kumugo S, Lunguya-Metila O, Mbenzo-Abokome V, Muyembe-Tamfum J, Delaporte E, Peeters M. High prevalence and a wide genetic diversity of simian retroviruses in non-human primate bushmeat in rural areas of the Democratic Republic of Congo. Ecohealth (in press))

Heigele A, Kmiec D, Regensburger K, Langer S, Peiffer L, Stürzel CM, Sauter D, Peeters M, Pizzato M, Learn GH, Hahn BH, Kirchhoff F.The Potency of Nef-Mediated SERINC5 Antagonism Correlates with the Prevalence of Primate Lentiviruses in the Wild. Cell Host Microbe. 2016;20(3):381-91.

Moeller AH, Caro-Quintero A, Mjungu D, Georgiev AV, Lonsdorf EV, Muller MN, Pusey AE, Peeters M, Hahn BH, Ochman H. Cospeciation of gut microbiota with hominids. Science. 2016;353(6297):380-2..

Liu W, Sundararaman SA, Loy DE, Learn GH, Li Y, Plenderleith LJ, Ndjango JB, Speede S, Atencia R, Cox D, Shaw GM, Ayouba A, Peeters M, Rayner JC, Hahn BH, Sharp PM. Multigenomic Delineation of Plasmodium Species of the Laverania Subgenus Infecting Wild-Living Chimpanzees and Gorillas. Genome Biol Evol. 2016;8(6):1929-39..

Sundararaman SA, Plenderleith LJ, Liu W, Loy DE, Learn GH, Li Y, Shaw KS, Ayouba A, Peeters M, Speede S, Shaw GM, Bushman FD, Brisson D, Rayner JC, Sharp PM, Hahn BH. Genomes of cryptic chimpanzee Plasmodium species reveal key evolutionary events leading to human malaria. Nat Commun. 2016;7:11078.

D’arc M, Ayouba A, Esteban A, Learn GH, Boué V, Liegeois F, Etienne L, Tagg N, Leendertz F, Boesch C, Madinda N, Robbins M, Gray M, Cournil A, Ooms M, Letko M, Simon V, Sharp P, Hahn B, Delaporte E, Mpoudi Ngole E, Peeters M. Origin of the HIV-1 group O epidemic in western lowland gorillas. Proc. Natl. Acad. Sci. USA. 2015 ; 112: E1343-52

Boué V, Locatelli S, Boucher F, Butel C, Esteban A, Okouga A, Ndoungouet A, Motsch P, Le Flohic G, Ngari P, Prugnolle P, Ollomo B, Rouet  F, Liégeois F. High Rate of Simian Immunodeficiency Virus (SIV) Infections in wild Living Chimpanzees in Northeastern Gabon. Viruses. 2015 Sep 15;7(9):4997-5015.

Villabona-Arenas CJ, Domyeum J, Mouacha F, Butel C, Delaporte E, Peeters M, Mpoudi-Ngole E, Aghokeng AF. HIV-1 group O infection in Cameroon from 2006 to 2013: Prevalence, genetic diversity, evolution and public health challenges. Infect Genet Evol. 2015; 36:210-216.

Ayouba A, Njouom R, Chia JE, Ahuka-Mundeke S, Kfutwah A, Ngole EM, Nerrienet E, Delaporte E, Peeters M. Molecular characterization of a new mosaic Simian Immunodeficiency Virus in a naturally infected tantalus monkey (Chlorocebus tantalus) from Cameroon: a challenge to the virus-host co-evolution of SIVagm in African green monkeys. Infect Genet Evol. 2015; 30:65-73.

Liégeois F, Schmidt F, Boué V, Butel C, Mouacha F, Ngari P, Ondo BM, Leroy E, Heeney JL, Delaporte E, Peeters M, Rouet F. Full-length genome analyses of two new simian immunodeficiency virus (SIV) strains from mustached monkeys (C. Cephus) in Gabon illustrate a complex evolutionary history among the SIVmus/mon/gsn lineage. Viruses. 2014 Jul 22;6(7):2880-98.

Gogarten JF, Akoua-Koffi C, Calvignac-Spencer S, Leendertz SA, Weiss S, Couacy-Hymann E, Koné I, Peeters M, Wittig RM, Boesch C, Hahn BH, Leendertz FH. The ecology of primate retroviruses - an assessment of 12 years of retroviral studies in the Taï national park area, Côte d׳Ivoire. Virology. 2014; 460-461:147-53.

Kluge S, Mack K, Iyer S, Pujol F, Heigele A, Learn G, Usmani S, Sauter D, Joas S, Hotter D, Bibollet-Ruche F, Plenderleith L, Peeters M, Geyer M, Sharp P, Fackler O, Hahn B, Kirchhoff, F. Nef proteins of epidemic HIV-1 group O strains antagonize human tetherin. Cell Host Microbe 2014 ; 16: 639-650

Moeller A, Peeters M, Ayouba A, Mpoudi Ngole E, Esteban A, Hahn B, Ochman H. Stability of the gorilla microbiome despite simian immunodeficiency virus infection. Mol. Ecol. 2015 ; 24:690-697

Moeller A, Li Y, Mpoudi Ngole E, Ahuka-Mundeke S, Lonsdorf E, Pusey A, Peeters M, Hahn B, Ochman H. Rapid changes in the gut microbiome during human evolution. Proc. Natl. Acad. Sci. USA. 2014 ; 111:16431-5

Mitchell MW, Locatelli S, Ghobrial L, Pokempner AA, Sesink Clee PR, Abwe EE, Nicholas A, Nkembi L, Anthony NM, Morgan BJ, Fotso R, Peeters M, Hahn BH, Gonder MK.The population genetics of wild chimpanzees in Cameroon and Nigeria suggests a positive role for selection in the evolution of chimpanzee subspecies. BMC Evol Biol. 2015;15:3.

Bittar F, Keita MB, Lagier JC, Peeters M, Delaporte E, Raoult D. Gorilla gorilla gorilla gut: a potential reservoir of pathogenic bacteria as revealed using culturomics and molecular tools. Sci Rep. 2014; 4:7174.

Fünfstück T, Arandjelovic M, Morgan DB, Sanz C, Reed P, Olson SH, Cameron K, Ondzie A, Peeters M, Vigilant L. The sampling scheme matters: Pan troglodytes troglodytes and P. t. schweinfurthii are characterized by clinal genetic variation rather than a strong subspecies break. Am J Phys Anthropol. 2015 Feb;156(2):181-91.

Hamad I, Keita MB, Peeters M, Delaporte E, Raoult D, Bittar F. Pathogenic eukaryotes in gut microbiota of western lowland gorillas as revealed by molecular survey. Sci Rep. 2014;4:6417.

Hamad I, Forestier CL, Peeters M, Delaporte E, Raoult D, Bittar F. Wild gorillas as a potential reservoir of Leishmania major. J Infect Dis. 2015;211(2):267-73.

Drakulovski P, Bertout S, Locatelli S, Butel C, Pion S, Mpoudi-Ngole E, Delaporte E, Peeters M, Mallié M. Assessment of gastrointestinal parasites in wild chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon. Parasitol Res. 2014;113(7):2541-50.

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