Investigation of caliciviruses and astroviruses in Gabonese rodents: A possible influence of national and international trade on the spread of enteric viruses.

Caliciviruses (Caliciviridae) and astroviruses (Astroviridae) are among the leading cause of non-bacterial foodborne disease and gastroenteritis in human. These non-enveloped RNA viruses infect a wide range of vertebrate species including rodents. Rodents are among the most important hosts of infectious diseases globally and are responsible for over 80 zoonotic pathogens that affect humans. Therefore, screening pathogens in rodents will be is necessary to prevent cross-species transmission to prevent zoonotic outbreaks. In the present study, we screened caliciviruses and astroviruses in order to describe their diversity and whether they harbor strains that can infect humans. RNA was then extracted from intestine samples of 245 rodents and retrotranscribed in cDNA to screen caliciviruses and astroviruses by PCRs. All the samples tested negative for caliciviruses and while astroviruses were detected in 18 (7.3%) samples of Rattus rattus species. Phylogenetic analyses based on the RdRp gene showed that all the sequences belonged to Mamastrovirus genus in which they were genetically related to R. rattus related AstVs previously detected in Gabon or in Rattus spp. AstV from Kenya and Asia. These findings suggested that transportation such as land and railway, as well national and international trade, are likely to facilitate spread of AstVs by the dissemination of rodents.

Absence of Coronavirus RNA in Faecal Samples from Wild Primates in Gabon, Central Africa.

Coronaviruses (CoVs, Coronaviridae) are a diverse group of viruses that infect mammals, birds, and fish. Seven CoVs infect humans, among which Severe Acute Respiratory Syndrome CoVs-1 and -2 and Middle East respiratory syndrome CoVs have shown how they can impact global health and the economy. Their spillover from bats-the natural reservoir-to humans has required intermediary hosts. Prevention requires that active surveillance be conducted on animals. Today, there is no data concerning the genetic diversity of CoVs naturally circulating in wild primates. This study aimed to screen wild great apes and mandrills in Gabon for CoVs. A total of 229 faecal samples of great apes and mandrills collected from 2009 to 2012 in forests and national parks were used for the detection of CoVs by nested PCR using primers targeting a conserved region of the RNA-dependent RNA polymerase. While all samples were negative, this lack of detection could be related to sample size, the transient nature of the infection, or because faecal samples are not suitable for detecting CoVs in primates. A longitudinal study should be performed and other non-invasive methods used to collect respiratory samples to better evaluate the circulation of CoVs in these primates.

Detection of Plasmodium falciparum in Saliva and Stool Samples from Children Living in Franceville, a Highly Endemic Region of Gabon.

Due to the difficulty of obtaining blood samples, which is the invasive method that is currently used for the detection of Plasmodium spp., alternative diagnostic sampling methods that are effective and non-invasive are needed, particularly for long-term studies. Saliva and stool samples from malaria-infected individuals contain trace amounts of Plasmodium DNA and therefore could be used as alternatives. Malaria was screened using rapid diagnosis tests and confirmed via microscopy. Nested PCR tests targeting the Plasmodium falciparum-specific STEVOR gene were performed for blood, saliva and stool samples that were positive for malaria. Three hundred sixty-seven (367) children were enrolled and eighty (22.22%) were confirmed to be positive for malaria. Matched blood, saliva and stool samples were available for 35 children. By using blood smears as the gold standard for the diagnosis of malaria, our study indicates that Plasmodium DNA was more detectable in blood (100%) than in saliva (22.86%) and stools (14.29%). Applying qPCR to the STEVOR gene to detect Plasmodium falciparum DNA in saliva and stool samples cannot be considered as an alternative to the current malaria detection processes using blood specimens.

Population Genomic Evidence of Adaptive Response during the Invasion History of Plasmodium falciparum in the Americas.

Plasmodium falciparum, the most virulent agent of human malaria, spread from Africa to all continents following the out-of-Africa human migrations. During the transatlantic slave trade between the 16th and 19th centuries, it was introduced twice independently to the Americas where it adapted to new environmental conditions (new human populations and mosquito species). Here, we analyzed the genome-wide polymorphisms of 2,635 isolates across the current P. falciparum distribution range in Africa, Asia, Oceania, and the Americas to investigate its genetic structure, invasion history, and selective pressures associated with its adaptation to the American environment. We confirmed that American populations originated from Africa with at least two independent introductions that led to two genetically distinct clusters, one in the North (Haiti and Colombia) and one in the South (French Guiana and Brazil), and an admixed Peruvian group. Genome scans revealed recent and more ancient signals of positive selection in the American populations. Particularly, we detected positive selection signals in genes involved in interactions with hosts (human and mosquito) cells and in genes involved in resistance to malaria drugs in both clusters. Analyses suggested that for five genes, adaptive introgression between clusters or selection on standing variation was at the origin of this repeated evolution. This study provides new genetic evidence on P. falciparum colonization history and on its local adaptation in the Americas.

Molecular Identification of Trypanosome Diversity in Domestic Animals Reveals the Presence of Trypanosoma brucei gambiense in Historical Foci of Human African Trypanosomiasis in Gabon.

Human African Trypanosomiasis (HAT) is an infectious disease caused by protozoan parasites belonging to the Trypanosoma genus. In sub-Saharan Africa, there is a significant threat as many people are at risk of infection. Despite this, HAT is classified as a neglected tropical disease. Over the last few years, several studies have reported the existence of a wide diversity of trypanosome species circulating in African animals. Thus, domestic and wild animals could be reservoirs of potentially dangerous trypanosomes for human populations. However, very little is known about the role of domestic animals in maintaining the transmission cycle of human trypanosomes in central Africa, especially in Gabon, where serious cases of infection are recorded each year, sometimes leading to hospitalization or death of patients. Komo-Mondah, located within Estuaries (Gabonese province), stays the most active HAT disease focus in Gabon, with a mean of 20 cases per year. In this study, we evaluated the diversity and prevalence of trypanosomes circulating in domestic animals using the Polymerase Chain Reaction (PCR) technique. We found that 19.34% (53/274) of the domestic animals we studied were infected with trypanosomes. The infection rates varied among taxa, with 23.21% (13/56) of dogs, 16.10% (19/118) of goats, and 21.00% (21/100) of sheep infected. In addition, we have observed a global mixed rate of infections of 20.75% (11/53) among infected individuals. Molecular analyses revealed that at least six Trypanosome species circulate in domestic animals in Gabon (T. congolense, T. simiae, T. simiae Tsavo, T. theileri, T. vivax, T. brucei (including T. brucei brucei, and T. brucei gambiense)). In conclusion, our study showed that domestic animals constitute important potential reservoirs for trypanosome parasites, including T. brucei gambiense, which is responsible for HAT.

Using haematophagous fly blood meals to study the diversity of blood-borne pathogens infecting wild mammals.

Many emerging infectious diseases originate from wild animals, so there is a profound need for surveillance and monitoring of their pathogens. However, the practical difficulty of sample acquisition from wild animals tends to limit the feasibility and effectiveness of such surveys. Xenosurveillance, using blood-feeding invertebrates to obtain tissue samples from wild animals and then detect their pathogens, is a promising method to do so. Here, we describe the use of tsetse fly blood meals to determine (directly through molecular diagnostic and indirectly through serology), the diversity of circulating blood-borne pathogens (including bacteria, viruses and protozoa) in a natural mammalian community of Tanzania. Molecular analyses of captured tsetse flies (182 pools of flies totalizing 1728 flies) revealed that the blood meals obtained came from 18 different vertebrate species including 16 non-human mammals, representing approximately 25% of the large mammal species present in the study area. Molecular diagnostic demonstrated the presence of different protozoa parasites and bacteria of medical and/or veterinary interest. None of the six virus species searched for by molecular methods were detected but an ELISA test detected antibodies against African swine fever virus among warthogs, indicating that the virus had been circulating in the area. Sampling of blood-feeding insects represents an efficient and practical approach to tracking a diversity of pathogens from multiple mammalian species, directly through molecular diagnostic or indirectly through serology, which could readily expand and enhance our understanding of the ecology and evolution of infectious agents and their interactions with their hosts in wild animal communities.

Evolutionary history of Plasmodium vivax and Plasmodium simium in the Americas.

Malaria is a vector-borne disease caused by protozoan parasites of the genus Plasmodium. Plasmodium vivax is the most prevalent human-infecting species in the Americas. However, the origins of this parasite in this continent are still debated. Similarly, it is now accepted that the existence of Plasmodium simium is explained by a P. vivax transfer from humans to monkey in America. However, many uncertainties still exist concerning the origin of the transfer and whether several transfers occurred. In this review, the most recent studies that addressed these questions using genetic and genomic approaches are presented.

A population genetic perspective on the origin, spread and adaptation of the human malaria agents Plasmodium falciparum and Plasmodium vivax.

Malaria is considered one of the most important scourges that humanity has faced during its history, being responsible every year for numerous deaths worldwide. The disease is caused by protozoan parasites, among which two species are responsible of the majority of the burden, Plasmodium falciparum and Plasmodium vivax. For these two parasite species, the questions of their origin (how and when they appeared in humans), of their spread throughout the world, as well as how they have adapted to humans have long been of interest to the scientific community. In this paper we review the existing body of knowledge, including current research dealing with these questions, focusing particularly on genetic and genomic analyses of these parasites and comparison with related Plasmodium species infecting other species of host (such as non-human primates).

Population genomic evidence of Plasmodium vivax Southeast Asian origin.

Plasmodium vivax is the most common and widespread human malaria parasite. It was recently proposed that P. vivax originates from sub-Saharan Africa based on the circulation of its closest genetic relatives (P. vivax-like) among African great apes. However, the limited number of genetic markers and samples investigated questions the robustness of this hypothesis. Here, we extensively characterized the genomic variations of 447 human P. vivax strains and 19 ape P. vivax-like strains collected worldwide. Phylogenetic relationships between human and ape Plasmodium strains revealed that P. vivax is a sister clade of P. vivax-like, not included within the radiation of P. vivax-like By investigating various aspects of P. vivax genetic variation, we identified several notable geographical patterns in summary statistics in function of the increasing geographic distance from Southeast Asia, suggesting that P. vivax may have derived from a single area in Asia through serial founder effects.

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents.

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.

Natural infection of free-ranging mandrills (Mandrillus sphinx) by enteroviruses and astroviruses in southern Gabon.

Enteroviruses (Picornaviridae) and astroviruses (Astroviridae) cause various diseases in humans and animals, including in non-human primates (NHPs). Some enteroviruses and astroviruses detected in NHPs are genetically related to those infecting humans, indicating the occurrence of interspecies transmissions. In this study, we screened 200 fecal samples of 56 free-ranging mandrills (Mandrillus sphinx) by nested reverse transcription-PCR with primers targeting the VP1 and RdRp genes, to evaluate the diversity of enterovirus and astrovirus infection, respectively, and the associated zoonotic risk. Overall, ten samples from six mandrills were enterovirus-positive (5%), and three samples from three mandrills were astrovirus-positive (1.5%). This is the first evidence of astrovirus infection in mandrills. Phylogenetic analyses based on the VP1 sequences revealed that all ten enterovirus sequences were part of the species Enterovirus J, suggesting low zoonotic risk. Phylogenetic analysis of the three astrovirus sequences showed that they all belonged to the Mamastrovirus genus. Two astrovirus sequences were highly divergent from all human astrovirus sequences (63.4-73% nucleotide identity), while one sequence (AstV-5) suggested cross-species transmission from humans to mandrills. Additional studies are needed to better characterize the identified astroviruses and to confirm whether mandrills are host of astroviruses than can be transmitted to humans.

A Second Genome Sequence of an Enterovirus C99 Detected in a Healthy Chimpanzee.

We report the nearly complete genome sequence of an enterovirus 99 strain (Cpz-IJC08) detected in a healthy chimpanzee from the Tchimpounga Sanctuary in the Republic of Congo. According to the phylogeny, Cpz-IJC08 clustered with Cpz-IJC04, a previously identified chimpanzee enterovirus from the same sanctuary, isolated from an animal with signs of acute flaccid paralysis.

Author Correction: Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human Plasmodium vivax diversity, population structure and evolutionary origin.

More than 200 million malaria clinical cases are reported each year due to Plasmodium vivax, the most widespread Plasmodium species in the world. This species has been neglected and understudied for a long time, due to its lower mortality in comparison with Plasmodium falciparum. A renewed interest has emerged in the past decade with the discovery of antimalarial drug resistance and of severe and even fatal human cases. Nonetheless, today there are still significant gaps in our understanding of the population genetics and evolutionary history of P. vivax, particularly because of a lack of genetic data from Africa. To address these gaps, we genotyped 14 microsatellite loci in 834 samples obtained from 28 locations in 20 countries from around the world. We discuss the worldwide population genetic structure and diversity and the evolutionary origin of P. vivax in the world and its introduction into the Americas. This study demonstrates the importance of conducting genome-wide analyses of P. vivax in order to unravel its complex evolutionary history.

The enigmatic mechanisms by which Plasmodium vivax infects Duffy-negative individuals.

The absence of the Duffy protein at the surface of erythrocytes was considered for decades to confer full protection against Plasmodium vivax as this blood group is the receptor for the key parasite ligand P. vivax Duffy binding protein (PvDBP). However, it is now clear that the parasite is able to break through this protection and induce clinical malaria in Duffy-negative people, although the underlying mechanisms are still not understood. Here, we briefly review the evidence of Duffy-negative infections by P. vivax and summarize the current hypothesis at the basis of this invasion process. We discuss those in the perspective of malaria-elimination challenges, notably in African countries.

Rodent malaria in Gabon: Diversity and host range.

Malaria parasites infect a wide range of vertebrate hosts, such as reptiles, birds and mammals (i.e., primates, ungulates, bats, and rodents). Four Plasmodium species and their subspecies infect African Muridae. Since their discoveries in the 1940s, these rodent Plasmodium species have served as biological models to explore many aspects of the biology of malaria agents and their interactions with their hosts. Despite that, surprisingly, little is known about their ecology, natural history and evolution. Most field studies on these parasites, performed from the 1940s to the early 1980s, showed that all rodent Plasmodium species infect only one main host species, the thicket rat. In the present study, we re-explored the diversity of Plasmodium parasites infecting rodent species living in peridomestic habitats in Gabon, Central Africa. Using molecular approaches, we found that at least two Plasmodium species (Plasmodium vinckei and Plasmodium yoelii) circulated among five rodent species (including the invasive species Mus musculus). This suggests that the host range of these parasites might be larger than previously considered. Our results also showed that the diversity of these parasites could be higher than currently recognized, with the discovery of a new phylogenetic lineage that could represent a new species of rodent Plasmodium.

Detection of novel astroviruses among rodents of Gabon, Central Africa.

Astroviruses (AstVs) are mostly responsible for mild to severe gastroenteritis infections in humans and animals. AstVs infect a wide range of host species, have a large genetic diversity with different circulating variants and are thus a high zoonotic risk for human populations. Among these host species, rodents are known to harbor several AstVs variants. Therefore, it is important to identify in rodent species which AstVs are circulating and evaluate their potential zoonotic risk for humans. In this context, this study aimed to screen the presence of AstVs in 267 rodents trapped in 2012 in Franceville and Makokou, two cities in Gabon. RNA extracted from grinded intestines were used for the screening of AstVs by amplification of a conserved region of the RNA dependent RNA polymerase. Results report the identification of AstVs in 12 individuals (4.6% rate), belonging to three different species including Rattus rattus, Mus musculus and Hybomys univittatus. These findings report the first identification of AstVs in R. rattus and H. univittatus. The phylogenetic analyses indicate host specificity of rodents AstVs. The absence of rodent AstVs within the human AstV clade suggests a low rate of interspecies transmission of these viruses and consequently a low zoonotic risk.

Extensive diversity of malaria parasites circulating in Central African bats and monkeys.

The order Haemosporidia gathers many protozoan parasites which are known to infect many host species and groups. Until recently, the studies on haemosporidian parasites primarily focused on the genus Plasmodium among a wide range of hosts. Genera, like the genus Hepatocystis, have received far less attention. In the present study, we present results of a survey of the diversity of Hepatocystis infecting bats and monkeys living in a same area in Gabon (Central Africa). Phylogenetic analyses revealed a large diversity of Hepatocystis lineages circulating among bats and monkeys, among which certain were previously observed in other African areas. Both groups of hosts harbor parasites belonging to distinct genetic clades and no transfers of parasites were observed between bats and monkeys. Finally, within each host group, no host specificity or geographical clustering was observed for the bat or the primate Hepatocystis lineages.