Final Report Summary - STRONGER (Strengthening transdisciplinary research on infectious and emerging diseases in French Guiana: linking fieldwork, benchside and bedside)
Executive Summary:
French Guiana (FG) is the sole EU outermost region in South America. It is also the largest forest for the EU. This tropical rain forest covers 90% of French Guiana (84 000 km2) and is a unique natural heritage in Europe with a high floral and faunistic diversity but fragile and vulnerable. Indeed, FG has to face important anthropogenic pressures. The population is estimated to be of 232 223 inhabitants, with 90% of the population living within the 3 main cities. The FG population is booming with a birth rate of 29.9 p. 1000, 3.7 children per woman, and 44.3% of the population below 20 years old. The annual mean growth rate is + 3.9% and the population is expected to have doubled in 2030. Moreover, this population booming is also due to a highly positive migratory balance.The economical growth is over 4%/year.
According to this context, FG presents all conditions to become a hot spot for emergence phenomena of infectious diseases in the future: low management of ecosystems facing important anthropogenic pressures, timber harvest, deforestation and fragmentation, conversion to agriculture lands result in ecological perturbations of wild fauna communities’ vertebrates as reservoirs and invertebrates as vectors and/or reservoirs. Moreover, the equatorial climate highly conduces to the multiplication of pathogens and is compatible with active reproduction of arthropod vectors. The increased contacts between human populations and their equatorial environment, the unplanned urbanization, strong immigration flux, the significant selection pressure for drug resistance ... are all risk factors favouring emergence phenomena. These conditions, in turn, expose human beings to wild cycles and favour the breakdown of species barriers. Indeed, over the last decades, FG already experienced several emergence phenomena : the first case of yellow fever in 1998 since 1902, the first human case of rabies in 2008, the first case of hantavirus pulmonary syndrome identified in 2008, the endemo-epidemicity of malaria, the hyper-endemicity of dengue fever, the HIV epidemic, the outbreaks of Chagas disease or Q fever ... The insufficiently well-known reasons of these emergence phenomena in humans required to create a scientific dynamic in the field of infectious and emerging diseases. To better understand the health risks posed by this growing interface between forest and human populations, Guianese research teams of the Institut Pasteur de la Guyane (IPG), the Centre National de la Recherche Scientifique (CNRS-UMR EcoFoG), the Institut National de la Santé et de la Recherche Médicale (INSERM-Centre d’investigation clinique – épidémiologie clinique, CIC - EC), the Université des Antilles et de la Guyane (Epidemiologie des Parasitoses Tropicales or EPAT) as well as several teams from continental Europe (The Netherlands, Portugal, United Kingdom) were associated in the STRonGer consortium.
STRonGer for " Strengthening transdisciplinary research on Infectious and Emerging Diseases in French Guiana: linking fieldwork, benchside and bedside" was a program whose objective was to significantly reinforce the existing medical research capacities in French Guiana - both in terms of human resources and infrastructure - to better meet the infectious health risks for the Guianese population.
As coordination and support actions, STRonGer has created a favourable context allowing the beneficiaries in French Guiana to be in the position to carry out research with the following strategic objectives:
- promoting a close cooperation and twinning actions for upgrading and boosting human potential in sciences and technologies;
- reinforcing diagnostic and management capacities by updating the available equipment and introducing new technologies;
- increasing the research capacities through highly qualified human resources and technical equipment;
- holding seminars and a scientific conference involving international experts to raise the local human capacities;
- disseminating the acquired expertise through different supports.
Project Context and Objectives:
STRonGer for " Strengthening transdisciplinary research on Infectious and Emerging Diseases in French Guiana: linking fieldwork, benchside and bedside" is a program whose objective was to significantly reinforce the existing medical research capacities in FG - both in terms of human resources and infrastructure - to better meet the infectious health risks for the Guianese population.
As coordination and support actions, STRonGer aimed at creating a favourable context allowing the beneficiaries in FG to be in the position to carry out research with the following strategic objectives:
‐ Promoting a close cooperation and twinning actions for upgrading and boosting human potential in sciences and technologies;
‐ Reinforcing diagnostic and management capacities by updating the available equipment and introducing new technologies;
‐ Increasing the research capacities through highly qualified human resources and technical equipment;
‐ Holding seminars and a scientific conference involving international experts to raise the local human capacities;
‐ Disseminating the acquired expertise through different supports.
The STRonGer program officially began on November 1st, 2011. Operationally, the kick-off meeting was held on December 13th and 14th, 2011.
Activities began with the recruitment of the researchers and technical assistants. To ensure equal treatment for all employees hired on the project and to not exceed the program budget, it has been decided, in agreement with the executive board of the Institut Pasteur in Paris, to establish fixed-term contracts operated by the Pasteur Institute of French Guiana.
One major concern about recruitments was to be attractive enough with respect to talented candidates knowing the competition with universities worldwide. In consequence, a special effort has been put on the dissemination of Job offers. They have been widely published through specialized websites, collaborative networks, social networks and specialized press. Each laboratory has selected its own candidates based on CV and cover letter and has made interviews. After this first selection, candidates were presented to the Steering Committee for approval. As a result, for all positions, a total of 189 CV has been received and 8 high-qualified young researchers as well as 1 engineer in bio-informatics and 1 clinical research assistant have been hired. Signing contracts was spread between June 2012 (M8) and February 2013 (M16), with a delay of nine months for the last compared with the initial schedule (M7). This delay was due to the necessity to launch several successive calls for different proposals when they were unsuccessful, the requirement of scientific excellence being the main criterion for the selection.
The second main objective was to upgrade the scientific platforms that will support the research activities of the STRonGer consortium. One of these platforms was that of the medical entomology unit (MEU) through the rehabilitation of two buildings: the first one to host a biosafety level-3 (BSL3) laboratory, insectaries and offices and the second one, dedicated to insecticide manipulations and molecular biology. Besides, extension and/or adjustment of the capacities of 3 research labs (Immunology, Virus-Host Interactions - LIVH, Parasitology) were planned in a third building. Finally, the BSL3 of the Virology Lab located in a fourth building had to be adjusted. Moreover, equipment of mostly all research labs had to be upgraded.
All these objectives had to be met at mid-term of the program. This was not the case for 3 main reasons:
- Given the importance of the rehabilitation work, it has been necessary to ask for an external expertise to help the Project Support Team (PST) and Work Package (WP) leaders in coordinating the work to be done and to ensure its proper implementation. It appeared also necessary to do the work within a regulatory framework that meets both the European rules on competition and use of public funds. Finally, it was also necessary to call for control agencies for technical inspection, security and health protection. In regard to these needs, two tenders not foreseen in the original project were launched: one for a Prime Contractor (PC) and one for a Technical assistance to the project owner (TAPO). The selection of beneficiaries was done according to the rules of public procurement (specifications, competitive bidding, publication, establishment of a Commission of tender). Moreover, a control agency was selected for technical inspection and security and health protection. All this process took over 3 months not initially planned;
- After the diagnosis phase carried out by the PC, it was concluded that the building scheduled to be renovated to accommodate the MEU BSL3 was not suited for a rehabilitation process. It was therefore necessary to review the entire MEU work program. The consortium decided to restore and extend another non occupied larger building located on the IPG campus to house all the MEU platforms, including the BSL3 lab, insectaries, insecticides and molecular biology labs, and offices. The elaboration of the new project, including final plans and work permit took over 5 months;
- Finally, the cost of the new project exceeded the initial budget and these extra costs had to be resolved before starting work. For that, it has been asked to each team of the consortium to redefine and update its equipment needs to reduce capital expenditures. Other funding solutions were sought, and a research program submitted to the European Regional Development Fund (ERDF) will allow IPG to finance the adjustment of the Virology Lab; finally, credit transfers within work packages were proposed and validated by the European project officer (EPO) on December 2012.
During the first period of the program (ie from November 1st 2011 to April 31 2013), 21 scientific research projects in connection with the STRonGer program were carried out by the consortium members; 9 were submitted to call for proposals and 3 formulation projects were in progress. Two participations of STRonGer members have been financed to an international Workshop in La Paz, Bolivia. The objective was to establish a Latin American network of collaboration among researchers on the theme of "Evolutionary molecular and genetic epidemiology of infectious diseases in Latin America”. Three scientific seminars have been organized in Cayenne, gathering a total of almost 80 researchers. The mid-term meeting took place from April 15th to 18th: more than 70 people participated to the opening conference, 21 international scientists were present and nine workshops have been organized.
Although some difficulties had to be dealt with during the first period, a real acceleration could be noticed during the second period of the STRonGer program (from May 1st 2013 to October 31st 2014) and all objectives were met by the end of October 2014.
After the mid-term meeting, 7 seminars were organized, 9 scientists have benefited from a scientific exchange in a partner institution abroad and 13 participations to international conferences were financed by the STRonGer program. This increased visibility at an international level of research activities in FG and led to new partnerships and projects which will be described later on (see page 44). The first “Amazonian Conference on Emerging and Infectious Diseases” boosted these objectives by gathering high qualified scientists from all over the world during a three-days event in Cayenne.
The upgrading of the scientific platform and of equipments was fully achieved during this second period. More than 1 000m2 of laboratories were renovated or created including a biosafety level-3 laboratory of 90m2. This high tech platform will allow settlement of new projects and boost cooperation with neighbor countries.
Thanks to the STRonGer program, the number of researchers in FG doubled. Among the 10 young scientists recruited within the framework of STRonGer, 5 will continue their research activities in French Guiana or the French West Indies within the Pasteur network. The dynamic induced by the STRonGer researchers had an impact at the regional level and local authorities decided to strengthen the scientific workforce in the next few years (recruitment of about 35 post-docs budgeted within the next 7 years) with the support of the ERDF.
Finally, the results of the STRonGer program, in terms of scientific activities and in terms of EU support, were largely disseminated through different communication tools. The recruitment of a part-time communication officer for the second period allowed a better coordination of dissemination activities. Since May 2013, eight video clips were issued and downloaded on dedicated YouTube channel and Facebook page, two brochures were edited and the didactic book dedicated to the identification of small Amazonian mammals in a context of emerging diseases was published.
According to the PO recommendations, the evaluation process did not start on the mid-term meeting as initially proposed but later on, once STRonGer activities were settled. Three visits were organized for the evaluators (September 2014, December 2014 and March 2015). The final report was transmitted to the project coordinator on April 8th 2015 and forwarded to the European Commission on April 9th and accepted on April 24th 2015.
All objectives listed in the Grant agreement were met by the end of April 2015.
Project Results:
Vector borne and emerging diseases
Thanks to the reinforcement of the scientific platform allowed by the STRonGer program, and particularly the creation of the Amazonian Vectopole, IPG will be able to participate to greater scientific projects on vector borne and emerging diseases thematics. As a matter of fact, IPG now has a double leading position:
‐ It is directly located on the field of emerging diseases and constitutes a European door with direct access to all kinds of biological materials;
‐ It has a leading-edge BSL3 laboratory and insectariums to allow thorough studies on vectors.
Thanks to seminars and meetings organized within the STRonGer program, a transdisciplinary dynamic has grown-up and several projects involving different teams could emerge.
Thereafter, it has been decided to work on the following three major axes to build-up wider transdisciplinary scientific projects involving both local teams and international partners:
‐ Vectors and arboviruses
‐ Vectors and malaria
‐ Biodiversity, reservoirs and emergence
Several brainstorming meetings have already taken place between teams to define project lines. These projects will then be presented to the EU and other partners for financial support.
Mosquito borne arboviroses in the Americas
All arboviruses originate from zoonotic cycle and spill off into human environment where urban mosquitoes take over transmission among vertebrates. While some viruses are now well installed in this environment (ex: Dengue virus, DENV, or chikungunya virus, CHIKV), some are still maintained in a selvatic cycle (ex: Mayaro (MAYV), Yellow Fever virus (YFV), West Nile virus (WNV)…..). These viruses threat to emerge, to expand at larger scale or have already conquered continents.
For the last decades, the Americas have faced this reality. Various countries of South America are experiencing more frequent, more intense and more severe outbreaks of DENV. WNV had first reached North America in 1999 and had spread out across the continent. The incidence of this disease amongst humans is however heterogeneous across its range. North American countries remain the most affected. More recently, CHIKV hit the Caribbean region and is currently spreading all over the continent.
In addition, South America is covered by large areas of tropical forests and other natural ecosystems creating ideal conditions for the occurrence of additional emerging and re-emerging arboviruses: MAYV, YFV, Saint Louis encephalitis virus (SLEV), Venezuelan Equine Encephalitis virus complex (VEEV). These viruses are circulating silently and are responsible for focal outbreaks or isolated cases.
These diseases are studied but gaps still need to be filled, and the inevitability of arboviral diseases overcome. Circulating virus strains are being characterized; vaccine development is undergoing for some of them; diagnosis tools exist but are still limited. Vectors are searched and identified among mosquito species; their ecology is being studied; vector control efficacy is being tested; but vector competence and its epidemiological reality are poorly documented. Human factors of severity are also searched for to improve health care but the role of pauci/asymptomatic in viral circulation needs to be investigated……
We have then decided to fill four main gaps by an integrative and multicentric approach across the American continent.
Our first objective is to improve diagnosis tools and cover a panel of putative circulating viruses. Accurate diagnosis is today a challenge because initial clinical symptoms of most arboviral diseases are common and can be confused with other infectious diseases that co-circulate in different regions of the Americas, complicating the clinical diagnosis. It is essential that a differential diagnosis be made to adopt appropriate clinical management. Reliable diagnosis tools are also necessary to develop strong research programs.
Our second objective is to unravel the factors favoring arbovirus circulation in the urban environment during inter-epidemic episodes and investigate the selvatisation of CHIKV in the Americas. A focus will be done on DENV, CHIKV and TONV. Our approach will integrate virus genotyping, vector capacity of mosquito species including the measure of vector competence, evaluation of human susceptibility and the role of pauci/asymptomatic as well as their behavior.
Our third objective is to evaluate which factors could modulate insecticide resistance phenotypes and vector competence for CHIKV, and eventually WNV. Insecticide resistance limits vector control efficacy and underlying mechanisms could also trigger pleiotropic effect modulating vector competence. Microbiota is also able to modulate vector competence and insecticide resistance. The investigation of interaction among these partners should give keys for understanding intraspecific vector competence modulation and disease dispersion.
Our fourth objective is to model the transmission of CHIKV to understand its spread, to characterize the short-term spatial dependence between cases and to identify the determinants of the virus transmission.
Scientific evidence for a better malaria control strategy in the Amazon region
For many years, several teams from the Institut Pasteur de la Guyane conducted research programs on malaria. The ambition of this strategic research project is to: i) bring together teams, means, and partners and, ii) coordinate research activities around the specificities of our territory in order to be able to clarify the situation in many other endemic areas in South America, but also in the World.
French Guiana is an Amazon region where malaria endemicity is low, transmitted by a particular vector which is very difficult to colonize, Anopheles darlingi, and where the epidemiological profiles are diverse. In addition, the Amazon region is historically an area of emergence regarding antimalarial drug resistances, usually concomitantly with the emergences observed in South East Asia. With the sharp decline in the number of malaria cases in the world linked to the use of artemisinin derivatives and a large distribution of impregnated mosquito nets, malaria elimination of falciparum cases is more than ever at the agenda of many national programs of many countries.
In this context, our objectives are:
- To enhance our knowledge on the mechanisms of resistance to artemisinin combination therapies (ACT) and more particularly, to understand the impact of artemisinin resistance on the resistance to its partner drug and vice versa. We will pay particular attention to artemisinin, lumefantrine, pyronaridine and piperaquine molecules that are the effective and usable drugs in the Amazon region. Resistant parasites will be identified: i) during the longitudinal monitoring carried out as a regular public health activity by our National Reference Center for malaria and WHO collaborating center activities or, ii) generated by in vitro drug pressure on natural isolates from French Guiana, recently adapted to in vitro multiplication.
- To understanding the evolution of malaria parasites in a context of low transmission and according to the sequential drug pressures applied on Plasmodium population. First, a general description of the genetic diversity and parasite dynamics across the department and the sub region would be done based on neutral markers of polymorphism. Secondly, this project aims to better understand the evolution of resistant parasites following changes in drug pressure and therefore switch in drug phenotypes (analysis of parasite fitness, time of emergence of revertant parasites, analysis of their dispersion at the continental scale etc ...).
- To raise Anopheles darlingi in the level 3 insectary available on site. This tool will help to address many questions on the transmission (vector competence depending on the susceptibility to insecticides or parasitic profiles, impact of infection on the aggressiveness of females...). Transmission blocking is a major point in the current elimination strategies. However, few antimalarial drugs act on the sexual stages of the parasite. The objective here is also to develop an in vitro test with which we will be able to evaluate the inhibition capacities of new compounds.
- To characterize malaria transmission in the field, in French Guiana, where we find the most infected people. This characterization will address: i) to present vectors and ii) to asymptomatic parasite carriers to compare the profile of parasites present in one or in the other but also ii) environmental factors influencing or possibly to predict the transmission level.
The ultimate goals of these four research lines intend to help governments to adjust their control/elimination programs based on scientific evidence.
Research axis on “Biodiversity, reservoirs and emergence”
Despite significant progress in the control and treatment of infectious diseases, bacteria, parasites, and viruses still remain the first cause of death in humans. The emergence of infectious diseases has become an important public health issue during the last four decades, especially in tropical areas. As already evoked, in the Amazonian region, disturbances related to increasing interfaces between human and the natural habitats, by modifying diversity, abundance, ecology, and dynamics of vectors and hosts, play an important role in the incidence of most viral diseases. These disorders include (i) strictly human factors such as population movements and uncontrolled sylvatic activities (ii) destruction of natural habitats: deforestation, fragmentation, uncontrolled urbanization, conversion of forest in agricultural lands. To understand, control and prevent the emergence of viral diseases, respective roles and importance of these pressures have to be assessed.
Looking for viruses
In the frame of the ViRUSES project funded by the European Regional Development Fund (FEDER/ERDF) for the 2009-2011 period, we were interested in understanding the emergence of several viral diseases related to interactions between wildlife, growing human populations and human-induced changes in habitats. Three viral families, responsible of rabies, arenavirus hemorrhagic fever or Hantavirus cardiopulmonary syndrome, have been investigated. Our choice took into consideration factors likely facilitating emergence phenomena: contact with forest habitats and vectors of viruses, urbanization, conversion of forest areas into urban and/or agricultural zones.
Trapping sessions of wild animals have been done on different sites and different environments in French Guiana. More than 1000 small terrestrial mammals and 1200 bats have been captured. These captures were used to characterize animal populations potentially reservoirs. Also, blood samples were taken and laboratory analyses realized to investigate the presence of these viruses and to study the dynamics of infection in different species over time and across sites.
Identification of new viruses
This project has allowed identifying through classical approaches two novel viruses (one Hantavirus and one Arenavirus) and their potential rodent reservoirs. The identification of human cases infected with the newly recognized Maripa hantavirus and of its associated reservoirs allowed to characterize areas and environmental conditions favorable to presence of rodents and thus potential areas of viral emergence. The identification of a new arenavirus in a rodent species, in a forest area near an agricultural zone, has allowed developing molecular tools to monitor its circulation in wild reservoirs.
Rabies virus circulation in bat populations
Information on the distribution and circulation of rabies virus in French Guiana were sparse. Detection of rabies virus in wild populations of bats demonstrated its wide circulation in several species (frugivorous, insectivorous and vampires) and its occurrence in numerous places. These findings have important implications as they suggest that the virus can be dispersed widely among bat populations. Nevertheless, only one vampire bat was found to be infective at the moment of its capture suggesting that the risk of transmission remains weak. While the number of viruses described in these animals (rodents and bats) increases every day, mechanisms associated with their maintenance and modes of transmission in wild populations remain poorly investigated. This gap of knowledge is striking for rabies virus for which bats are considered the main reservoir in South America but for which only few data are available on the virus circulation in time and space and on its mortality or morbidity rates. All these data highlighted the importance of pursuing this work to better understand the mechanisms of emergence/maintenance of these viruses.
Dynamics of the hosts and their viruses
The pursuit of our trapping effort first allows collecting extensive data over time on the distribution and dynamics of wild mammal communities. As for example, in the frame of the CAROLIA project funded by the ERDF/FEDER funds for the 2012-2015 period, weperform a spatial and temporal monitoring of rabies in different bat species, especially on vampire bats. These animals are captured, sampled for blood, individually marked and released. Capture/Mark/recapture in two specific roots allows estimating the population size and dispersal ability of vampires and of their associated viruses, as well as exchanges between populations. This monitoring will also allow gaining a better understanding of the ecological and epidemiological factors that could explain the emergence and dispersal of this virus. Modes of transmission, duration of seroconversion in vampires but also period during which the virus is excreted by these animals will allow to better understand how the virus is maintained in the host and dispersed. At last, genetic studies on bats are ongoing using different types of markers (MHC class II, microsatellites, Cytochrome b). They will help identifying the population structure of the three studied bat species (Desmodus rotundus, Carollia perspicilliata and Molossus molossus).
From classical tools to metagenomic approaches for virus identification
In addition, we collaborated, within STRonGer, with Ernst J. Verschoor (European partner) from the Biomedical Primate Research Centre, The Netherlands, on a program dedicated to virus discovery in bats. By specifically targeting the Polyomaviridae family we have been able to characterize different viruses from 8 distinct species. To enlarge our search on emerging viruses in bat populations, a study of the viral diversity using a metagenomic approach has been initiated. Christiane Bouchier from the Plateforme Génomique, Pasteur Institute, brought her expertise in next generation sequencing approaches and helped us to initiate collaborations with the bioinformatics team at IP. Meanwhile, thanks to STRonGer, a bioinformatician has been hired. This allowed us to develop several projects using metagenomic and genomic approaches that are dedicated to the characterization of viral biodiversity in mammal reservoir species (Virome and BioViRo, funded by LabEx CEBA). While Virome is dedicated to the characterization of the viral diversity in three different bat species, the objective of BioViro is to characterize viral diversity in several species of rodents collected in various environments including habitats close to human settlements. This project should allow identifying new viruses potentially pathogenic for humans and characterizing the infectious environment to which human communities (traditional and modern) may have to face.
Genomics of adaptation
Using similar approaches we have developed studies on the evolutionary/genetic processes of adaptation of the host to viral infections (Amazomics, funded by Labex CEBA). The purpose of this project is to address the question of adaptation genomics in two different bat species (Sturnira lilium and Molossus molossus). These two species have been selected due to their presence in two distinct ecosystems (Martinique vs. French Guiana) and are thus suspected to have evolved under different environmental and parasitic pressures. This provides a unique opportunity to test respective importance of ecological, biological and pathogen constraints in these two species by looking for genotype/environment (virome) associations.
Immune response of bats to viral infection
In parallel to the population genetics and viral metagenomic studies conducted on bats, we thought that it was essential to understand the immune mechanisms involved in the early recognition of viral infection. The description of different markers of innate immunity, corresponding to the first line of defense of the host, such as Toll-like receptors and RIG-I like receptors, has been initiated in the frame of CAROLIA and thanks to the hiring of a post-doc within STRonGer. This allowed us first to characterize different genes (TLR-3, -7, -8, -9, RIG-I, OAS-1, ...) from three different New World bat species that differ significantly from each other based on their diet (hematophagous, insectivorous and frugivorous) and second to analyze their expression in different organs. As a follow-up of this initial study, we have set-up a research project called BATIMMUNE with Eliane Meurs, head of the Hepacivirus unit at the Pasteur Institute, funded by the Pasteur Institute through a Transversal Research Program (PTR 499) for the 2015-2016 period. An important point of the program is to investigate how the different characterized genes are engaged in the innate immune response of bats in stress conditions such as viral infection. To do so, we will establish immortalized cell lines from different organs of the three bat species and submit them to chemical treatments or even viral infection. For this purpose we will benefit from the new BSL-3 facility in which we have a dedicated room. We will be able to evaluate the efficacy of the bat pathogen recognition receptors and identify the downstream signaling pathways. Use of immortalized cell lines will permit to generate robust and reproducible data. Moreover, construction of reporter bat cell lines containing the luciferase gene under the control of the identified IFN promoter or of the IFN-responsive promoter will be extremely useful to follow-up induction of IFN and response to IFN in the bat cell lines upon stimulation/infection and, in particular, for further programs on infection with rabies virus. All these aspects should give us insights on the infectious process and response to infection of bats.
Potential Impact:
Cooperation activities
Parasitology Laboratory & UMR EcoFoG
During the STRonGer program the parasitology lab and UMR EcoFog investigated the Plasmodium falciparum sexual stage, responsible for Malaria transmission from infected patients to mosquitoes. The main objective was to set-up a pipeline of protocols to identify drugs that could act against sexual stages of Plasmodium falciparum. The asexual (symptomatic-responsible) blood stage bioassay is already routinely used at IPG; no bioassay targeting sexual stages is however available.
To acquire experience in this field, we established a new collaboration with the group of Robert Sinden at Imperial College (London, UK). Sinden’s team is among the world leaders in Plasmodium gametocytogenesis and also in transmission-blocking bioassays for drug development. Jean Popovici (STRonGer postdoctoral associate) spent 3 weeks in Sinden’s group and completed a training to acquire the expertise to perform such bioassay. He transfered the methods back to IPG.
Nowadays, the method is back to the lab and some experiments are conducted in order to evaluate the activity of several food dyes. The implementation of this method apply to recently adapted malaria field isolates from French Guiana will be publish soon.
Parasitology laboratory
The laboratory works on drug resistance in parasites responsible for malaria, either P. falciparum or P. vivax. Its activities are mainly focused on topics of main interest in public health in our Amazonian region. In order to strengthen its links with regional teams, it initiated collaborations with the foundation of tropical diseases in Manaus and more precisely, Dr Marcus Lacerda, which is an expert in clinical studies and P. vivax malaria. It is now collaborating on two projects: i) evaluating the expense of chloroquine drug resistance reversion and artemisinin resistance in the Amazonian region based on molecular marker studies ; ii) evaluating the therapeutic efficacy of artemisinin combination therapies through a multicentric clinical studies also implicating a Surinamese team. The initiation of the collaboration was mainly facilitated by the STRonGer program, which financed a seminar given by Dr Marcus Lacerda in French Guiana. From that entry point, collaboration on dengue is discussed with the virology lab.
The development of high throughput methods of genotyping offers large possibilities to better understand mechanisms of resistance in malaria parasites. These methods require also highly skilled people in bioinformatics. The bioinformatics unit of the IPG being at its very beginning, we have chosen to collaborate with a team located in Cambridge, MA, USA, at the Broad Institute specialized in Plasmodium sequencing and its specificity, an highly AT rich genome (90%). Dr Dan Neafsey, a researcher in population genetics has been invited to give a seminar on the whole genome sequencing methods and their applications in medical sciences. From there, a strong collaboration has been initiated between the two teams that are really complementary, one in the cutting edge technologies and the other one in the field, in transmission area. This collaboration is up to now really fruitful with one publication submitted on the genetic diversity of malaria parasites and another one in preparation on the first identification in the field, of reversal mechanism in resistance of malaria parasites to drugs. The STRonGer program has initiated that collaboration by giving financial support to: i) Dan Neafsey for his trip to French Guiana for a seminar and, ii) Lise Musset and Stéphane Pelleau for a training at the Broad Institute on analyzing data from high throughput sequencing data.
A second person working with Dan from the Harvard School of Public Health, Sarah Volkman, gave a video-conference talk during the ACEID conference in September 2014.
Virology
The STRonGer program enabled us to invite Marco Vignuzzi from the Institut Pasteur, Paris. Following his lecture on « Chikungunya : emergence et transmission d’intermédiaires évolutifs à potentiel épidémique » in February 2014, 2 workshops have been conducted, leading to the establishment of collaborations with his team.
A first collaboration involves the IPG Virology (D Rousset and Epidemiology units (C Flamand), as well as the Infectious and Tropical Diseases Unit (Dr F Djossou) at Cayenne Hospital together with IPP Viral Populations and Pathogenesis Unit (M Vignuzzi) around the DENGUE–OMIC project. This project funded by LabEx CEBA funds, aims to study viral quasispecies diversity in severe dengue infection cases that occurred during the 2013 dengue epidemic in French Guiana
Virology and MEU
Another collaboration involves at IPG the Medical Entomology Unit (I Dusfour) andVirology Unit (D Rousset), the Oswaldo Cruz Institute (Ricardo Lourenço de Oliveira) at Rio de Janeiro, the Virology Unit (V Duong) at IP of Cambodia with IPP Viral Populations and Pathogenesis Unit (M Vignuzzi) around the PTR 489 CHIKAlert. This research program focuses on mosquito and human driven evolution of chikungunya virus in the Americas and on the identification on future adaptative intermediates.
The STRonGer program also enabled IPG Virology Unit to strengthen collaboration with the CIBU (Cellule d’Intervention Biologique d’Urgence, IPP) through the training of Sophie De Decker (post doctoral researcher –Virology Unit IPG) to NGS and primary sample pretreatment analysis for the identification of pathogens using Next Generation Sequencing methods. Once back in Cayenne, Sophie presented the competences she had acquired to the Virology team.
Medical Entomology
The STRonGer program enabled the IPG Medical Entomology Unit to develop collaborations with:
- Joao Pinto and his team (Institute of Hygiene and Tropical Medicine, Lisbon). J. Pinto (STRonGer partner present at KOM in December 2011 and ACEID in September 2014) and P. Salgueiro (present at ACEID) are now partners in a research program entitled SPREAD. This program focuses on the dynamics of insecticide resistance in the mosquito Aedes aegypti. It is co-funded by the IP and FioCruz, co-led by I. Dusfour (MEU - IPG) and AJ. Martins (FioCruz). The latter also attended the ACEID. This collaboration has also enabled the welcome of I. Dusfour within the team of J. Pinto for a 15 days training course to genetic studies of mosquitoes populations.
- Lisa Randford-Cartwright and her team (University of Glasgow). L. Randford- Cartwright (STRonGer partner, present at KOM in December 2011, MTM in April 2013 and ACEID in September 2014) hosted in her laboratory S. Vezenegho (IPG - MEU, post-doctorant), for the completion of a 6 weeks training to experimental infection studies of malaria mosquitoes. A research program, integrating work based on this new skill, is currently submitted to the National Research Agency (ANR) for funding. - Ralph Harbach (Natural History Museum, London). R. Harbach (STRonGer partner, present at MTM in April 2013) provided support for taxonomic work of MEU.
- Jean-Philippe David and team (LECA, Grenoble). J-P. David (invited to give a seminar in November 2013) and F. Falcon (attended the ACEID in September 2014) are now partners in a research program called REAGIR. This program aims to evaluate new insecticide candidates for the fight against the Aedes aegypti mosquito. It is funded by ANSES and managed by I. Dusfour (IPG - MEU).
- Patrick Mavingui (University of Lyon). P. Mavingui (present in ACEID in September 2014) is now a partner of one of the strategic research programs under development at IPG.
- Marta Moreno and team (University of California San Diego). M. Moreno (invited to give a seminar in October 2014) now provides support for the establishment of anopheline mosquitoes’ colony in the Vectopôle insectary.
CIC EC and EPaT
The CIC 1424 and the EA3593 benefited from the STRonGer project through fundings allowing to invite to Cayenne molecular epidemiology of Histoplasma capsulatum specialist Dr Zancope from Fiocruz in Rio de Janeiro. This first visit took place during the midterm meeting and allowed to look for research goals. There was exchange of histoplasma DNA to start exploring different methods.
Subsequently, with PAHO support, a team from the CIC and from the EA3593 went to Fiocruz to further the collaboration. Since then a PhD student has started working on the topic thanks to a French Ministry of Overseas Territories’ grant. STRonGer also enabled Infectiologist and Mycologist Pr Olivier Lortholary’s mission, to give a seminar in Cayenne. Collaborations continue with regular exchanges and the project of looking at galactomanan on the samples from a study on the diagnosis of histoplasmosis in HIV infected patients (ANRS, ERDF funds).
During the midterm meeting Pr Mark Wainberg was also invited to give a talk on the molecular epidemiology of HIV. The research has not started yet because of regulatory delays.
At the EPAT at last, Aurelia Stefani (STRonGer post doc) was involved in collaborating with Amapa State researchers regarding remote sensing and malaria mapping (Guyamapa).
LIVH
The STRonGer program enabled IPG Virus-Host Interactions Laboratory (IPG - LIVH) to develop collaborations with:
- Ernst J. Verschoor and team (Biomedical Primate Research Centre – The Netherlands). E. J. Verschoor (STRonGer European partner) attended STRonGer KOM in December 2011 and MTM in April 2013. He collaborates with LIVH team on a project dedicated to virus discovery in bats. This collaboration led to identification of new polyomaviruses in several bat species from French Guiana. The results have been published in 2012 (Fagrouch Z et al. 2012). Furthermore, both laboratories
(BPRC and LIVH), working for a decade on primate herpesviruses, have collaborated on the characterization of novel roseoloviruses in African great apes close to human herpesvirus 7. This work led to a publication in 2014 (Lavergne A et al. 2014).
- Eliane Meurs and team (Hepacivirus Unit - Institut Pasteur, Paris). E. Meurs (invited to the MTM in April 2013) welcomed Marie-Claude Lise (IPG – LIVH, postdoc) in her laboratory in June 2014 for a six-weeks training aiming at producing lentiviral particles to generate immortalized bat cell lines. E. Meurs brings her expertise on innate immune system and is now partner of LIVH in a research program called BATIMMUNE funded by IPP in the frame of a Transversal Research Program (PTR 499). This program aims to better understand bat’s innate immune response to viral infection.
- Christiane Bouchier and team (Plateforme Génomique - Institut Pasteur, Paris). C. Bouchier was invited at IPG in October 2012 to give a seminar on “Next Generation Sequencing approaches”. She brings her expertise in this domain and helped the LIVH to initiate collaborations with the bioinformatics team at IP. She is now a LIVH partner in several projects using metagenomic and genomic approaches, that are dedicated to the characterization of viral biodiversity in mammal reservoir species or microbes (Virome, BioViRo, Genevil funded by LabEx CEBA) and of adaptation of the host to viral infections (Amazomics funded by Labex CEBA).
- Alvaro Aguilar-Sétien and team (IMSS - Mexico). A. Aguilar-Sétien was invited to the MTM in April 2013.He gave a talk on projects developed by his lab on experimental infection of bats (Artibeus sp.) with rabies or dengue viruses. A. Aguilar Sétien collaborates with the LIVH on a project studying the genetic structure of different vampire bat communities from French Guiana, Brazil and Mexico (CAROLIA project funded by ERDF). Furthermore, a collaboration is initiated on infection of bat species. Members of LIVH have to go to Mexico to test immunological tools developed at IPG on bats (Artibeus species) infected with rabies virus. This project was submitted to a Mexican fund.
List of Websites:
http://www.pasteur-cayenne.fr/stronger/
French Guiana (FG) is the sole EU outermost region in South America. It is also the largest forest for the EU. This tropical rain forest covers 90% of French Guiana (84 000 km2) and is a unique natural heritage in Europe with a high floral and faunistic diversity but fragile and vulnerable. Indeed, FG has to face important anthropogenic pressures. The population is estimated to be of 232 223 inhabitants, with 90% of the population living within the 3 main cities. The FG population is booming with a birth rate of 29.9 p. 1000, 3.7 children per woman, and 44.3% of the population below 20 years old. The annual mean growth rate is + 3.9% and the population is expected to have doubled in 2030. Moreover, this population booming is also due to a highly positive migratory balance.The economical growth is over 4%/year.
According to this context, FG presents all conditions to become a hot spot for emergence phenomena of infectious diseases in the future: low management of ecosystems facing important anthropogenic pressures, timber harvest, deforestation and fragmentation, conversion to agriculture lands result in ecological perturbations of wild fauna communities’ vertebrates as reservoirs and invertebrates as vectors and/or reservoirs. Moreover, the equatorial climate highly conduces to the multiplication of pathogens and is compatible with active reproduction of arthropod vectors. The increased contacts between human populations and their equatorial environment, the unplanned urbanization, strong immigration flux, the significant selection pressure for drug resistance ... are all risk factors favouring emergence phenomena. These conditions, in turn, expose human beings to wild cycles and favour the breakdown of species barriers. Indeed, over the last decades, FG already experienced several emergence phenomena : the first case of yellow fever in 1998 since 1902, the first human case of rabies in 2008, the first case of hantavirus pulmonary syndrome identified in 2008, the endemo-epidemicity of malaria, the hyper-endemicity of dengue fever, the HIV epidemic, the outbreaks of Chagas disease or Q fever ... The insufficiently well-known reasons of these emergence phenomena in humans required to create a scientific dynamic in the field of infectious and emerging diseases. To better understand the health risks posed by this growing interface between forest and human populations, Guianese research teams of the Institut Pasteur de la Guyane (IPG), the Centre National de la Recherche Scientifique (CNRS-UMR EcoFoG), the Institut National de la Santé et de la Recherche Médicale (INSERM-Centre d’investigation clinique – épidémiologie clinique, CIC - EC), the Université des Antilles et de la Guyane (Epidemiologie des Parasitoses Tropicales or EPAT) as well as several teams from continental Europe (The Netherlands, Portugal, United Kingdom) were associated in the STRonGer consortium.
STRonGer for " Strengthening transdisciplinary research on Infectious and Emerging Diseases in French Guiana: linking fieldwork, benchside and bedside" was a program whose objective was to significantly reinforce the existing medical research capacities in French Guiana - both in terms of human resources and infrastructure - to better meet the infectious health risks for the Guianese population.
As coordination and support actions, STRonGer has created a favourable context allowing the beneficiaries in French Guiana to be in the position to carry out research with the following strategic objectives:
- promoting a close cooperation and twinning actions for upgrading and boosting human potential in sciences and technologies;
- reinforcing diagnostic and management capacities by updating the available equipment and introducing new technologies;
- increasing the research capacities through highly qualified human resources and technical equipment;
- holding seminars and a scientific conference involving international experts to raise the local human capacities;
- disseminating the acquired expertise through different supports.
Project Context and Objectives:
STRonGer for " Strengthening transdisciplinary research on Infectious and Emerging Diseases in French Guiana: linking fieldwork, benchside and bedside" is a program whose objective was to significantly reinforce the existing medical research capacities in FG - both in terms of human resources and infrastructure - to better meet the infectious health risks for the Guianese population.
As coordination and support actions, STRonGer aimed at creating a favourable context allowing the beneficiaries in FG to be in the position to carry out research with the following strategic objectives:
‐ Promoting a close cooperation and twinning actions for upgrading and boosting human potential in sciences and technologies;
‐ Reinforcing diagnostic and management capacities by updating the available equipment and introducing new technologies;
‐ Increasing the research capacities through highly qualified human resources and technical equipment;
‐ Holding seminars and a scientific conference involving international experts to raise the local human capacities;
‐ Disseminating the acquired expertise through different supports.
The STRonGer program officially began on November 1st, 2011. Operationally, the kick-off meeting was held on December 13th and 14th, 2011.
Activities began with the recruitment of the researchers and technical assistants. To ensure equal treatment for all employees hired on the project and to not exceed the program budget, it has been decided, in agreement with the executive board of the Institut Pasteur in Paris, to establish fixed-term contracts operated by the Pasteur Institute of French Guiana.
One major concern about recruitments was to be attractive enough with respect to talented candidates knowing the competition with universities worldwide. In consequence, a special effort has been put on the dissemination of Job offers. They have been widely published through specialized websites, collaborative networks, social networks and specialized press. Each laboratory has selected its own candidates based on CV and cover letter and has made interviews. After this first selection, candidates were presented to the Steering Committee for approval. As a result, for all positions, a total of 189 CV has been received and 8 high-qualified young researchers as well as 1 engineer in bio-informatics and 1 clinical research assistant have been hired. Signing contracts was spread between June 2012 (M8) and February 2013 (M16), with a delay of nine months for the last compared with the initial schedule (M7). This delay was due to the necessity to launch several successive calls for different proposals when they were unsuccessful, the requirement of scientific excellence being the main criterion for the selection.
The second main objective was to upgrade the scientific platforms that will support the research activities of the STRonGer consortium. One of these platforms was that of the medical entomology unit (MEU) through the rehabilitation of two buildings: the first one to host a biosafety level-3 (BSL3) laboratory, insectaries and offices and the second one, dedicated to insecticide manipulations and molecular biology. Besides, extension and/or adjustment of the capacities of 3 research labs (Immunology, Virus-Host Interactions - LIVH, Parasitology) were planned in a third building. Finally, the BSL3 of the Virology Lab located in a fourth building had to be adjusted. Moreover, equipment of mostly all research labs had to be upgraded.
All these objectives had to be met at mid-term of the program. This was not the case for 3 main reasons:
- Given the importance of the rehabilitation work, it has been necessary to ask for an external expertise to help the Project Support Team (PST) and Work Package (WP) leaders in coordinating the work to be done and to ensure its proper implementation. It appeared also necessary to do the work within a regulatory framework that meets both the European rules on competition and use of public funds. Finally, it was also necessary to call for control agencies for technical inspection, security and health protection. In regard to these needs, two tenders not foreseen in the original project were launched: one for a Prime Contractor (PC) and one for a Technical assistance to the project owner (TAPO). The selection of beneficiaries was done according to the rules of public procurement (specifications, competitive bidding, publication, establishment of a Commission of tender). Moreover, a control agency was selected for technical inspection and security and health protection. All this process took over 3 months not initially planned;
- After the diagnosis phase carried out by the PC, it was concluded that the building scheduled to be renovated to accommodate the MEU BSL3 was not suited for a rehabilitation process. It was therefore necessary to review the entire MEU work program. The consortium decided to restore and extend another non occupied larger building located on the IPG campus to house all the MEU platforms, including the BSL3 lab, insectaries, insecticides and molecular biology labs, and offices. The elaboration of the new project, including final plans and work permit took over 5 months;
- Finally, the cost of the new project exceeded the initial budget and these extra costs had to be resolved before starting work. For that, it has been asked to each team of the consortium to redefine and update its equipment needs to reduce capital expenditures. Other funding solutions were sought, and a research program submitted to the European Regional Development Fund (ERDF) will allow IPG to finance the adjustment of the Virology Lab; finally, credit transfers within work packages were proposed and validated by the European project officer (EPO) on December 2012.
During the first period of the program (ie from November 1st 2011 to April 31 2013), 21 scientific research projects in connection with the STRonGer program were carried out by the consortium members; 9 were submitted to call for proposals and 3 formulation projects were in progress. Two participations of STRonGer members have been financed to an international Workshop in La Paz, Bolivia. The objective was to establish a Latin American network of collaboration among researchers on the theme of "Evolutionary molecular and genetic epidemiology of infectious diseases in Latin America”. Three scientific seminars have been organized in Cayenne, gathering a total of almost 80 researchers. The mid-term meeting took place from April 15th to 18th: more than 70 people participated to the opening conference, 21 international scientists were present and nine workshops have been organized.
Although some difficulties had to be dealt with during the first period, a real acceleration could be noticed during the second period of the STRonGer program (from May 1st 2013 to October 31st 2014) and all objectives were met by the end of October 2014.
After the mid-term meeting, 7 seminars were organized, 9 scientists have benefited from a scientific exchange in a partner institution abroad and 13 participations to international conferences were financed by the STRonGer program. This increased visibility at an international level of research activities in FG and led to new partnerships and projects which will be described later on (see page 44). The first “Amazonian Conference on Emerging and Infectious Diseases” boosted these objectives by gathering high qualified scientists from all over the world during a three-days event in Cayenne.
The upgrading of the scientific platform and of equipments was fully achieved during this second period. More than 1 000m2 of laboratories were renovated or created including a biosafety level-3 laboratory of 90m2. This high tech platform will allow settlement of new projects and boost cooperation with neighbor countries.
Thanks to the STRonGer program, the number of researchers in FG doubled. Among the 10 young scientists recruited within the framework of STRonGer, 5 will continue their research activities in French Guiana or the French West Indies within the Pasteur network. The dynamic induced by the STRonGer researchers had an impact at the regional level and local authorities decided to strengthen the scientific workforce in the next few years (recruitment of about 35 post-docs budgeted within the next 7 years) with the support of the ERDF.
Finally, the results of the STRonGer program, in terms of scientific activities and in terms of EU support, were largely disseminated through different communication tools. The recruitment of a part-time communication officer for the second period allowed a better coordination of dissemination activities. Since May 2013, eight video clips were issued and downloaded on dedicated YouTube channel and Facebook page, two brochures were edited and the didactic book dedicated to the identification of small Amazonian mammals in a context of emerging diseases was published.
According to the PO recommendations, the evaluation process did not start on the mid-term meeting as initially proposed but later on, once STRonGer activities were settled. Three visits were organized for the evaluators (September 2014, December 2014 and March 2015). The final report was transmitted to the project coordinator on April 8th 2015 and forwarded to the European Commission on April 9th and accepted on April 24th 2015.
All objectives listed in the Grant agreement were met by the end of April 2015.
Project Results:
Vector borne and emerging diseases
Thanks to the reinforcement of the scientific platform allowed by the STRonGer program, and particularly the creation of the Amazonian Vectopole, IPG will be able to participate to greater scientific projects on vector borne and emerging diseases thematics. As a matter of fact, IPG now has a double leading position:
‐ It is directly located on the field of emerging diseases and constitutes a European door with direct access to all kinds of biological materials;
‐ It has a leading-edge BSL3 laboratory and insectariums to allow thorough studies on vectors.
Thanks to seminars and meetings organized within the STRonGer program, a transdisciplinary dynamic has grown-up and several projects involving different teams could emerge.
Thereafter, it has been decided to work on the following three major axes to build-up wider transdisciplinary scientific projects involving both local teams and international partners:
‐ Vectors and arboviruses
‐ Vectors and malaria
‐ Biodiversity, reservoirs and emergence
Several brainstorming meetings have already taken place between teams to define project lines. These projects will then be presented to the EU and other partners for financial support.
Mosquito borne arboviroses in the Americas
All arboviruses originate from zoonotic cycle and spill off into human environment where urban mosquitoes take over transmission among vertebrates. While some viruses are now well installed in this environment (ex: Dengue virus, DENV, or chikungunya virus, CHIKV), some are still maintained in a selvatic cycle (ex: Mayaro (MAYV), Yellow Fever virus (YFV), West Nile virus (WNV)…..). These viruses threat to emerge, to expand at larger scale or have already conquered continents.
For the last decades, the Americas have faced this reality. Various countries of South America are experiencing more frequent, more intense and more severe outbreaks of DENV. WNV had first reached North America in 1999 and had spread out across the continent. The incidence of this disease amongst humans is however heterogeneous across its range. North American countries remain the most affected. More recently, CHIKV hit the Caribbean region and is currently spreading all over the continent.
In addition, South America is covered by large areas of tropical forests and other natural ecosystems creating ideal conditions for the occurrence of additional emerging and re-emerging arboviruses: MAYV, YFV, Saint Louis encephalitis virus (SLEV), Venezuelan Equine Encephalitis virus complex (VEEV). These viruses are circulating silently and are responsible for focal outbreaks or isolated cases.
These diseases are studied but gaps still need to be filled, and the inevitability of arboviral diseases overcome. Circulating virus strains are being characterized; vaccine development is undergoing for some of them; diagnosis tools exist but are still limited. Vectors are searched and identified among mosquito species; their ecology is being studied; vector control efficacy is being tested; but vector competence and its epidemiological reality are poorly documented. Human factors of severity are also searched for to improve health care but the role of pauci/asymptomatic in viral circulation needs to be investigated……
We have then decided to fill four main gaps by an integrative and multicentric approach across the American continent.
Our first objective is to improve diagnosis tools and cover a panel of putative circulating viruses. Accurate diagnosis is today a challenge because initial clinical symptoms of most arboviral diseases are common and can be confused with other infectious diseases that co-circulate in different regions of the Americas, complicating the clinical diagnosis. It is essential that a differential diagnosis be made to adopt appropriate clinical management. Reliable diagnosis tools are also necessary to develop strong research programs.
Our second objective is to unravel the factors favoring arbovirus circulation in the urban environment during inter-epidemic episodes and investigate the selvatisation of CHIKV in the Americas. A focus will be done on DENV, CHIKV and TONV. Our approach will integrate virus genotyping, vector capacity of mosquito species including the measure of vector competence, evaluation of human susceptibility and the role of pauci/asymptomatic as well as their behavior.
Our third objective is to evaluate which factors could modulate insecticide resistance phenotypes and vector competence for CHIKV, and eventually WNV. Insecticide resistance limits vector control efficacy and underlying mechanisms could also trigger pleiotropic effect modulating vector competence. Microbiota is also able to modulate vector competence and insecticide resistance. The investigation of interaction among these partners should give keys for understanding intraspecific vector competence modulation and disease dispersion.
Our fourth objective is to model the transmission of CHIKV to understand its spread, to characterize the short-term spatial dependence between cases and to identify the determinants of the virus transmission.
Scientific evidence for a better malaria control strategy in the Amazon region
For many years, several teams from the Institut Pasteur de la Guyane conducted research programs on malaria. The ambition of this strategic research project is to: i) bring together teams, means, and partners and, ii) coordinate research activities around the specificities of our territory in order to be able to clarify the situation in many other endemic areas in South America, but also in the World.
French Guiana is an Amazon region where malaria endemicity is low, transmitted by a particular vector which is very difficult to colonize, Anopheles darlingi, and where the epidemiological profiles are diverse. In addition, the Amazon region is historically an area of emergence regarding antimalarial drug resistances, usually concomitantly with the emergences observed in South East Asia. With the sharp decline in the number of malaria cases in the world linked to the use of artemisinin derivatives and a large distribution of impregnated mosquito nets, malaria elimination of falciparum cases is more than ever at the agenda of many national programs of many countries.
In this context, our objectives are:
- To enhance our knowledge on the mechanisms of resistance to artemisinin combination therapies (ACT) and more particularly, to understand the impact of artemisinin resistance on the resistance to its partner drug and vice versa. We will pay particular attention to artemisinin, lumefantrine, pyronaridine and piperaquine molecules that are the effective and usable drugs in the Amazon region. Resistant parasites will be identified: i) during the longitudinal monitoring carried out as a regular public health activity by our National Reference Center for malaria and WHO collaborating center activities or, ii) generated by in vitro drug pressure on natural isolates from French Guiana, recently adapted to in vitro multiplication.
- To understanding the evolution of malaria parasites in a context of low transmission and according to the sequential drug pressures applied on Plasmodium population. First, a general description of the genetic diversity and parasite dynamics across the department and the sub region would be done based on neutral markers of polymorphism. Secondly, this project aims to better understand the evolution of resistant parasites following changes in drug pressure and therefore switch in drug phenotypes (analysis of parasite fitness, time of emergence of revertant parasites, analysis of their dispersion at the continental scale etc ...).
- To raise Anopheles darlingi in the level 3 insectary available on site. This tool will help to address many questions on the transmission (vector competence depending on the susceptibility to insecticides or parasitic profiles, impact of infection on the aggressiveness of females...). Transmission blocking is a major point in the current elimination strategies. However, few antimalarial drugs act on the sexual stages of the parasite. The objective here is also to develop an in vitro test with which we will be able to evaluate the inhibition capacities of new compounds.
- To characterize malaria transmission in the field, in French Guiana, where we find the most infected people. This characterization will address: i) to present vectors and ii) to asymptomatic parasite carriers to compare the profile of parasites present in one or in the other but also ii) environmental factors influencing or possibly to predict the transmission level.
The ultimate goals of these four research lines intend to help governments to adjust their control/elimination programs based on scientific evidence.
Research axis on “Biodiversity, reservoirs and emergence”
Despite significant progress in the control and treatment of infectious diseases, bacteria, parasites, and viruses still remain the first cause of death in humans. The emergence of infectious diseases has become an important public health issue during the last four decades, especially in tropical areas. As already evoked, in the Amazonian region, disturbances related to increasing interfaces between human and the natural habitats, by modifying diversity, abundance, ecology, and dynamics of vectors and hosts, play an important role in the incidence of most viral diseases. These disorders include (i) strictly human factors such as population movements and uncontrolled sylvatic activities (ii) destruction of natural habitats: deforestation, fragmentation, uncontrolled urbanization, conversion of forest in agricultural lands. To understand, control and prevent the emergence of viral diseases, respective roles and importance of these pressures have to be assessed.
Looking for viruses
In the frame of the ViRUSES project funded by the European Regional Development Fund (FEDER/ERDF) for the 2009-2011 period, we were interested in understanding the emergence of several viral diseases related to interactions between wildlife, growing human populations and human-induced changes in habitats. Three viral families, responsible of rabies, arenavirus hemorrhagic fever or Hantavirus cardiopulmonary syndrome, have been investigated. Our choice took into consideration factors likely facilitating emergence phenomena: contact with forest habitats and vectors of viruses, urbanization, conversion of forest areas into urban and/or agricultural zones.
Trapping sessions of wild animals have been done on different sites and different environments in French Guiana. More than 1000 small terrestrial mammals and 1200 bats have been captured. These captures were used to characterize animal populations potentially reservoirs. Also, blood samples were taken and laboratory analyses realized to investigate the presence of these viruses and to study the dynamics of infection in different species over time and across sites.
Identification of new viruses
This project has allowed identifying through classical approaches two novel viruses (one Hantavirus and one Arenavirus) and their potential rodent reservoirs. The identification of human cases infected with the newly recognized Maripa hantavirus and of its associated reservoirs allowed to characterize areas and environmental conditions favorable to presence of rodents and thus potential areas of viral emergence. The identification of a new arenavirus in a rodent species, in a forest area near an agricultural zone, has allowed developing molecular tools to monitor its circulation in wild reservoirs.
Rabies virus circulation in bat populations
Information on the distribution and circulation of rabies virus in French Guiana were sparse. Detection of rabies virus in wild populations of bats demonstrated its wide circulation in several species (frugivorous, insectivorous and vampires) and its occurrence in numerous places. These findings have important implications as they suggest that the virus can be dispersed widely among bat populations. Nevertheless, only one vampire bat was found to be infective at the moment of its capture suggesting that the risk of transmission remains weak. While the number of viruses described in these animals (rodents and bats) increases every day, mechanisms associated with their maintenance and modes of transmission in wild populations remain poorly investigated. This gap of knowledge is striking for rabies virus for which bats are considered the main reservoir in South America but for which only few data are available on the virus circulation in time and space and on its mortality or morbidity rates. All these data highlighted the importance of pursuing this work to better understand the mechanisms of emergence/maintenance of these viruses.
Dynamics of the hosts and their viruses
The pursuit of our trapping effort first allows collecting extensive data over time on the distribution and dynamics of wild mammal communities. As for example, in the frame of the CAROLIA project funded by the ERDF/FEDER funds for the 2012-2015 period, weperform a spatial and temporal monitoring of rabies in different bat species, especially on vampire bats. These animals are captured, sampled for blood, individually marked and released. Capture/Mark/recapture in two specific roots allows estimating the population size and dispersal ability of vampires and of their associated viruses, as well as exchanges between populations. This monitoring will also allow gaining a better understanding of the ecological and epidemiological factors that could explain the emergence and dispersal of this virus. Modes of transmission, duration of seroconversion in vampires but also period during which the virus is excreted by these animals will allow to better understand how the virus is maintained in the host and dispersed. At last, genetic studies on bats are ongoing using different types of markers (MHC class II, microsatellites, Cytochrome b). They will help identifying the population structure of the three studied bat species (Desmodus rotundus, Carollia perspicilliata and Molossus molossus).
From classical tools to metagenomic approaches for virus identification
In addition, we collaborated, within STRonGer, with Ernst J. Verschoor (European partner) from the Biomedical Primate Research Centre, The Netherlands, on a program dedicated to virus discovery in bats. By specifically targeting the Polyomaviridae family we have been able to characterize different viruses from 8 distinct species. To enlarge our search on emerging viruses in bat populations, a study of the viral diversity using a metagenomic approach has been initiated. Christiane Bouchier from the Plateforme Génomique, Pasteur Institute, brought her expertise in next generation sequencing approaches and helped us to initiate collaborations with the bioinformatics team at IP. Meanwhile, thanks to STRonGer, a bioinformatician has been hired. This allowed us to develop several projects using metagenomic and genomic approaches that are dedicated to the characterization of viral biodiversity in mammal reservoir species (Virome and BioViRo, funded by LabEx CEBA). While Virome is dedicated to the characterization of the viral diversity in three different bat species, the objective of BioViro is to characterize viral diversity in several species of rodents collected in various environments including habitats close to human settlements. This project should allow identifying new viruses potentially pathogenic for humans and characterizing the infectious environment to which human communities (traditional and modern) may have to face.
Genomics of adaptation
Using similar approaches we have developed studies on the evolutionary/genetic processes of adaptation of the host to viral infections (Amazomics, funded by Labex CEBA). The purpose of this project is to address the question of adaptation genomics in two different bat species (Sturnira lilium and Molossus molossus). These two species have been selected due to their presence in two distinct ecosystems (Martinique vs. French Guiana) and are thus suspected to have evolved under different environmental and parasitic pressures. This provides a unique opportunity to test respective importance of ecological, biological and pathogen constraints in these two species by looking for genotype/environment (virome) associations.
Immune response of bats to viral infection
In parallel to the population genetics and viral metagenomic studies conducted on bats, we thought that it was essential to understand the immune mechanisms involved in the early recognition of viral infection. The description of different markers of innate immunity, corresponding to the first line of defense of the host, such as Toll-like receptors and RIG-I like receptors, has been initiated in the frame of CAROLIA and thanks to the hiring of a post-doc within STRonGer. This allowed us first to characterize different genes (TLR-3, -7, -8, -9, RIG-I, OAS-1, ...) from three different New World bat species that differ significantly from each other based on their diet (hematophagous, insectivorous and frugivorous) and second to analyze their expression in different organs. As a follow-up of this initial study, we have set-up a research project called BATIMMUNE with Eliane Meurs, head of the Hepacivirus unit at the Pasteur Institute, funded by the Pasteur Institute through a Transversal Research Program (PTR 499) for the 2015-2016 period. An important point of the program is to investigate how the different characterized genes are engaged in the innate immune response of bats in stress conditions such as viral infection. To do so, we will establish immortalized cell lines from different organs of the three bat species and submit them to chemical treatments or even viral infection. For this purpose we will benefit from the new BSL-3 facility in which we have a dedicated room. We will be able to evaluate the efficacy of the bat pathogen recognition receptors and identify the downstream signaling pathways. Use of immortalized cell lines will permit to generate robust and reproducible data. Moreover, construction of reporter bat cell lines containing the luciferase gene under the control of the identified IFN promoter or of the IFN-responsive promoter will be extremely useful to follow-up induction of IFN and response to IFN in the bat cell lines upon stimulation/infection and, in particular, for further programs on infection with rabies virus. All these aspects should give us insights on the infectious process and response to infection of bats.
Potential Impact:
Cooperation activities
Parasitology Laboratory & UMR EcoFoG
During the STRonGer program the parasitology lab and UMR EcoFog investigated the Plasmodium falciparum sexual stage, responsible for Malaria transmission from infected patients to mosquitoes. The main objective was to set-up a pipeline of protocols to identify drugs that could act against sexual stages of Plasmodium falciparum. The asexual (symptomatic-responsible) blood stage bioassay is already routinely used at IPG; no bioassay targeting sexual stages is however available.
To acquire experience in this field, we established a new collaboration with the group of Robert Sinden at Imperial College (London, UK). Sinden’s team is among the world leaders in Plasmodium gametocytogenesis and also in transmission-blocking bioassays for drug development. Jean Popovici (STRonGer postdoctoral associate) spent 3 weeks in Sinden’s group and completed a training to acquire the expertise to perform such bioassay. He transfered the methods back to IPG.
Nowadays, the method is back to the lab and some experiments are conducted in order to evaluate the activity of several food dyes. The implementation of this method apply to recently adapted malaria field isolates from French Guiana will be publish soon.
Parasitology laboratory
The laboratory works on drug resistance in parasites responsible for malaria, either P. falciparum or P. vivax. Its activities are mainly focused on topics of main interest in public health in our Amazonian region. In order to strengthen its links with regional teams, it initiated collaborations with the foundation of tropical diseases in Manaus and more precisely, Dr Marcus Lacerda, which is an expert in clinical studies and P. vivax malaria. It is now collaborating on two projects: i) evaluating the expense of chloroquine drug resistance reversion and artemisinin resistance in the Amazonian region based on molecular marker studies ; ii) evaluating the therapeutic efficacy of artemisinin combination therapies through a multicentric clinical studies also implicating a Surinamese team. The initiation of the collaboration was mainly facilitated by the STRonGer program, which financed a seminar given by Dr Marcus Lacerda in French Guiana. From that entry point, collaboration on dengue is discussed with the virology lab.
The development of high throughput methods of genotyping offers large possibilities to better understand mechanisms of resistance in malaria parasites. These methods require also highly skilled people in bioinformatics. The bioinformatics unit of the IPG being at its very beginning, we have chosen to collaborate with a team located in Cambridge, MA, USA, at the Broad Institute specialized in Plasmodium sequencing and its specificity, an highly AT rich genome (90%). Dr Dan Neafsey, a researcher in population genetics has been invited to give a seminar on the whole genome sequencing methods and their applications in medical sciences. From there, a strong collaboration has been initiated between the two teams that are really complementary, one in the cutting edge technologies and the other one in the field, in transmission area. This collaboration is up to now really fruitful with one publication submitted on the genetic diversity of malaria parasites and another one in preparation on the first identification in the field, of reversal mechanism in resistance of malaria parasites to drugs. The STRonGer program has initiated that collaboration by giving financial support to: i) Dan Neafsey for his trip to French Guiana for a seminar and, ii) Lise Musset and Stéphane Pelleau for a training at the Broad Institute on analyzing data from high throughput sequencing data.
A second person working with Dan from the Harvard School of Public Health, Sarah Volkman, gave a video-conference talk during the ACEID conference in September 2014.
Virology
The STRonGer program enabled us to invite Marco Vignuzzi from the Institut Pasteur, Paris. Following his lecture on « Chikungunya : emergence et transmission d’intermédiaires évolutifs à potentiel épidémique » in February 2014, 2 workshops have been conducted, leading to the establishment of collaborations with his team.
A first collaboration involves the IPG Virology (D Rousset and Epidemiology units (C Flamand), as well as the Infectious and Tropical Diseases Unit (Dr F Djossou) at Cayenne Hospital together with IPP Viral Populations and Pathogenesis Unit (M Vignuzzi) around the DENGUE–OMIC project. This project funded by LabEx CEBA funds, aims to study viral quasispecies diversity in severe dengue infection cases that occurred during the 2013 dengue epidemic in French Guiana
Virology and MEU
Another collaboration involves at IPG the Medical Entomology Unit (I Dusfour) andVirology Unit (D Rousset), the Oswaldo Cruz Institute (Ricardo Lourenço de Oliveira) at Rio de Janeiro, the Virology Unit (V Duong) at IP of Cambodia with IPP Viral Populations and Pathogenesis Unit (M Vignuzzi) around the PTR 489 CHIKAlert. This research program focuses on mosquito and human driven evolution of chikungunya virus in the Americas and on the identification on future adaptative intermediates.
The STRonGer program also enabled IPG Virology Unit to strengthen collaboration with the CIBU (Cellule d’Intervention Biologique d’Urgence, IPP) through the training of Sophie De Decker (post doctoral researcher –Virology Unit IPG) to NGS and primary sample pretreatment analysis for the identification of pathogens using Next Generation Sequencing methods. Once back in Cayenne, Sophie presented the competences she had acquired to the Virology team.
Medical Entomology
The STRonGer program enabled the IPG Medical Entomology Unit to develop collaborations with:
- Joao Pinto and his team (Institute of Hygiene and Tropical Medicine, Lisbon). J. Pinto (STRonGer partner present at KOM in December 2011 and ACEID in September 2014) and P. Salgueiro (present at ACEID) are now partners in a research program entitled SPREAD. This program focuses on the dynamics of insecticide resistance in the mosquito Aedes aegypti. It is co-funded by the IP and FioCruz, co-led by I. Dusfour (MEU - IPG) and AJ. Martins (FioCruz). The latter also attended the ACEID. This collaboration has also enabled the welcome of I. Dusfour within the team of J. Pinto for a 15 days training course to genetic studies of mosquitoes populations.
- Lisa Randford-Cartwright and her team (University of Glasgow). L. Randford- Cartwright (STRonGer partner, present at KOM in December 2011, MTM in April 2013 and ACEID in September 2014) hosted in her laboratory S. Vezenegho (IPG - MEU, post-doctorant), for the completion of a 6 weeks training to experimental infection studies of malaria mosquitoes. A research program, integrating work based on this new skill, is currently submitted to the National Research Agency (ANR) for funding. - Ralph Harbach (Natural History Museum, London). R. Harbach (STRonGer partner, present at MTM in April 2013) provided support for taxonomic work of MEU.
- Jean-Philippe David and team (LECA, Grenoble). J-P. David (invited to give a seminar in November 2013) and F. Falcon (attended the ACEID in September 2014) are now partners in a research program called REAGIR. This program aims to evaluate new insecticide candidates for the fight against the Aedes aegypti mosquito. It is funded by ANSES and managed by I. Dusfour (IPG - MEU).
- Patrick Mavingui (University of Lyon). P. Mavingui (present in ACEID in September 2014) is now a partner of one of the strategic research programs under development at IPG.
- Marta Moreno and team (University of California San Diego). M. Moreno (invited to give a seminar in October 2014) now provides support for the establishment of anopheline mosquitoes’ colony in the Vectopôle insectary.
CIC EC and EPaT
The CIC 1424 and the EA3593 benefited from the STRonGer project through fundings allowing to invite to Cayenne molecular epidemiology of Histoplasma capsulatum specialist Dr Zancope from Fiocruz in Rio de Janeiro. This first visit took place during the midterm meeting and allowed to look for research goals. There was exchange of histoplasma DNA to start exploring different methods.
Subsequently, with PAHO support, a team from the CIC and from the EA3593 went to Fiocruz to further the collaboration. Since then a PhD student has started working on the topic thanks to a French Ministry of Overseas Territories’ grant. STRonGer also enabled Infectiologist and Mycologist Pr Olivier Lortholary’s mission, to give a seminar in Cayenne. Collaborations continue with regular exchanges and the project of looking at galactomanan on the samples from a study on the diagnosis of histoplasmosis in HIV infected patients (ANRS, ERDF funds).
During the midterm meeting Pr Mark Wainberg was also invited to give a talk on the molecular epidemiology of HIV. The research has not started yet because of regulatory delays.
At the EPAT at last, Aurelia Stefani (STRonGer post doc) was involved in collaborating with Amapa State researchers regarding remote sensing and malaria mapping (Guyamapa).
LIVH
The STRonGer program enabled IPG Virus-Host Interactions Laboratory (IPG - LIVH) to develop collaborations with:
- Ernst J. Verschoor and team (Biomedical Primate Research Centre – The Netherlands). E. J. Verschoor (STRonGer European partner) attended STRonGer KOM in December 2011 and MTM in April 2013. He collaborates with LIVH team on a project dedicated to virus discovery in bats. This collaboration led to identification of new polyomaviruses in several bat species from French Guiana. The results have been published in 2012 (Fagrouch Z et al. 2012). Furthermore, both laboratories
(BPRC and LIVH), working for a decade on primate herpesviruses, have collaborated on the characterization of novel roseoloviruses in African great apes close to human herpesvirus 7. This work led to a publication in 2014 (Lavergne A et al. 2014).
- Eliane Meurs and team (Hepacivirus Unit - Institut Pasteur, Paris). E. Meurs (invited to the MTM in April 2013) welcomed Marie-Claude Lise (IPG – LIVH, postdoc) in her laboratory in June 2014 for a six-weeks training aiming at producing lentiviral particles to generate immortalized bat cell lines. E. Meurs brings her expertise on innate immune system and is now partner of LIVH in a research program called BATIMMUNE funded by IPP in the frame of a Transversal Research Program (PTR 499). This program aims to better understand bat’s innate immune response to viral infection.
- Christiane Bouchier and team (Plateforme Génomique - Institut Pasteur, Paris). C. Bouchier was invited at IPG in October 2012 to give a seminar on “Next Generation Sequencing approaches”. She brings her expertise in this domain and helped the LIVH to initiate collaborations with the bioinformatics team at IP. She is now a LIVH partner in several projects using metagenomic and genomic approaches, that are dedicated to the characterization of viral biodiversity in mammal reservoir species or microbes (Virome, BioViRo, Genevil funded by LabEx CEBA) and of adaptation of the host to viral infections (Amazomics funded by Labex CEBA).
- Alvaro Aguilar-Sétien and team (IMSS - Mexico). A. Aguilar-Sétien was invited to the MTM in April 2013.He gave a talk on projects developed by his lab on experimental infection of bats (Artibeus sp.) with rabies or dengue viruses. A. Aguilar Sétien collaborates with the LIVH on a project studying the genetic structure of different vampire bat communities from French Guiana, Brazil and Mexico (CAROLIA project funded by ERDF). Furthermore, a collaboration is initiated on infection of bat species. Members of LIVH have to go to Mexico to test immunological tools developed at IPG on bats (Artibeus species) infected with rabies virus. This project was submitted to a Mexican fund.
List of Websites:
http://www.pasteur-cayenne.fr/stronger/
