Search

Dengue virus (DENV). belonging to flavivirus genus (Flaviviridae family) is the etiologic agent of dengue disease which the most prevalent arboviral infection in humans. There are four antigenically different serotypes (DENV-1 to DENV-4) which are all transmitted to humans following the bite of female Aedes mosquitoes. More than half of the world's population is affected by dengue, and more particularly those living in the tropical and subtropical areas of the Americas, Asia and Oceania where the effective mosquito vectors have spread. The four serotypes of DENV serotypes can cause a wide spectrum of clinical manifestations which range from flu-like disease to dengue severe (or dengue hemorrhagic fever). Dengue became a concern in South West Indian Ocean (SWIO) region with an epidemic of DENV-2 in 2018 in La Reunion Island. The viral strain involved in epidemic La Reunion 2018 DENV-2 belongs to the “Cosmopolitan” genotype and was probably imported from the Seychelles. Little information is available on the biological characteristics of circulating “Cosmopolitan” DENV-2 strains in SWIO. In response to the DENV-2 epidemic of 2018, UMR PIMIT developed a complete study on clinical isolate RUN-18 viral strain which was isolated in Reunion. As part of my thesis project, I was more particularly interested with the non-structural glycoprotein NS1 of RUN-18 which contributes to viral RNA replication but which is also found released from infected cells as a soluble hexamer lipoprotein complexes. The secreted soluble form of NS1 may play a central role in dengue pathogenesis by disrupting the functions of the vascular endothelium. I developed a research project focused on the biological properties of NS1 RUN-18 protein using a recombinant protein rNS1 expressed in human hepatocyte Huh 7 cells. Expression of rNS1 RUN-18 was compared to rNS1 from DENV-2 DES-14 strain (Cosmopolitan genotype) that was isolated in Tanzania during an dengue outbreak in 2014. The major finding of my study was the weak expression level of rNS1 RUN-18 in Huh 7 cells as compared to rNS1 DES-14. Four amino acid changes at positions NS1-128/131/272/324 differentiate NS1 RUN-18 and NS1 DES-14. By site-directed mutagenesis, I demonstrated that the lysine residues at positions 272 and 324 may play a key role in the low level of expression and secretion of rNS1 RUN-18. The introduction of R272K and R324K substitutions in rNS1 DES-14 caused a marked reduction in protein expression level leading to a significant release of LDH associated with a decrease in mitochondrial metabolism, signatures of increased cytotoxicity. Analysis of rNS1 expression in Huh 7 cells revealed the ability of the protein to interact with the lipids in hepatocyte cells. Indeed, the soluble form of rNS1 can associate with apolipoproteins B and A1 produced in Huh7 cells. Furthermore, the secreted form of rNS1 has the ability to interact with endothelial cells leading to a marked decrease in the permeability of endothelial cell monolayers as well as an activation of inflammatory pathways associated with oxidative stress. My thesis project allowed to provide original information on biological properties of the NS1 protein from epidemic La Réunion 2018 DENV-2 strain.; La dengue est une arbovirose majeure en santé publique dont l’agent étiologique est le virus de la dengue (DENV) qui appartient au genre flavivirus (famille Flaviviridae). DENV est composé de 4 sérotypes (DENV-1 à DENV-4) qui sont tous transmis à l’homme suite à la piqûre par les moustiques du genre Aedes. Plus de la moitié de la population mondiale est concernée aujourd’hui par la dengue et plus particulièrement celles vivant dans les régions tropicales et subtropicales des Amériques, de l’Asie et de l’Océanie qui sont infestées par les moustiques vecteurs de la maladie. L’infection par les quatre sérotypes de DENV est responsable d’un large spectre de manifestations cliniques et certains individus développent une fièvre dengue qui peut aboutir à une dengue sévère (ou dengue hémorragique) pouvant entrainer la mort. La région du Sud-Ouest de l’océan Indien (SOOI) est concernée par la dengue avec une épidémie de DENV-2 qui a démarré en 2018 à La Réunion. La souche virale responsable de l’épidémie appartient au génotype « Cosmopolitan » et a été probablement importée des Seychelles. Peu d’informations sont disponibles sur les caractéristiques biologiques des DENV-2 « Cosmopolitan » présents dans le SOOI. En réponse à l’épidémie de DENV-2 de 2018, l’UMR PIMIT a lancé une étude générale sur la souche virale RUN-18 qui a été isolée à La Réunion. Dans le cadre de mon projet de thèse, je me suis plus particulièrement intéressée à la protéine non-structurale NS1 de RUN-18 qui participe à la réplication virale, mais qui est aussi retrouvée excrétée des cellules infectées sous la forme d’un hexamère associé aux lipides. La forme soluble sécrétée de NS1 contribue au pouvoir pathogène de DENV en perturbant les fonctions de l’endothélium vasculaire. J’ai développé un projet de recherche sur la caractérisation de la protéine NS1 RUN-18 produite sous la forme d’une protéine recombinante rNS1 exprimée dans les cellules hépatocytaires humaines Huh7. Une analyse comparative a été menée entre la rNS1 RUN-18 et celle de la souche épidémique de DENV-2 « Cosmopolitan » isolée en Tanzanie en 2014 (rNS1 DES-14). rNS1 RUN-18 est faiblement exprimée et sécrétée des cellules Huh7 en comparaison de ce qui est observé avec rNS1 DES-14. Quatre changements d’acides aminés aux positions NS1-128/131/272/324 différencient NS1 RUN-18 et DES-14. Par mutagenèse dirigée, j’ai démontré que les résidus lysine aux positions 272 et 324 jouent un rôle déterminant dans le faible niveau d’expression et de sécrétion de rNS1 RUN-18. L’introduction des substitutions R272K et R324K dans rNS1 DES-14 réduit fortement le niveau d’expression de la protéine dans les cellules Huh7 avec en parallèle une importante libération de LDH associée à une diminution du métabolisme mitochondrial, signatures d’une cytotoxicité accrue. Je me suis ensuite intéressée à l’interaction des rNS1 RUN-18 et DES-14 avec les lipides des cellules hépatocytaires. J’ai montré que la forme soluble de rNS1 peut s’associer aux apolipoprotéines B et A1 produites par les cellules Huh7. Aussi, la forme sécrétée de rNS1 a la capacité d’interagir avec les cellules endothéliales amenant à une diminution marquée de la perméabilité des monocouches de cellules endothéliales ainsi qu’une activation des voies de l’inflammation associé à un stress oxydatif. L’ensemble de mes travaux de thèse a permis d’apporter des informations originales sur la protéine NS1 de la souche épidémique Réunion 2018 DENV-2 en termes de données biochimiques, biologiques et physiopathologiques qui permettront de mieux comprendre le pouvoir pathogène des DENV-2 génotype « Cosmopolitan » retrouvées dans le SOOI.

Dengue is the most widespread arbovirosis in the world, with approximately 390 million cases per year, 96 millions of which have clinical manifestations and 25,000 deaths. In West Africa, the circulation of this virus in human populations was first reported in the 1960s in Nigeria. Clinical diagnosis of dengue in West Africa is made difficult by the existence of other diseases with similar clinical presentations. Biological diagnosis remains therefore the only alternative. This biological diagnosis requires high quality equipment and well-trained personnel, which are not always available in resource-limited countries. Thus, many cases of dengue fever are consistently reported as malaria, leading to mismanagement, which can have serious consequences on the health status of patients. It is therefore necessary to set up surveillance systems for febrile infections of unknown origin in Africa by strengthening the diagnostic capacities of national laboratories.Circulation du virus de la dengue en Afrique de l’Ouest - Une problématique émergente de santé publique.La dengue est l’arbovirose la plus répandue dans le monde avec environ 390 millions de cas par an, dont 96 millions présentent des manifestations cliniques, avec plus de 25 152 décès annuels répertoriés. Le diagnostic clinique de la dengue en Afrique de l’Ouest est rendu difficile par l’existence d’autres maladies présentant des tableaux cliniques similaires. Il est donc nécessaire de mettre en place des systèmes de surveillance des infections fébriles d’origine inconnue en Afrique, en renforçant les capacités diagnostiques des laboratoires nationaux.

Au cours de ces derni��res ann��es, la circulation de la dengue dans la r��gion Pacifique et en Nouvelle-Cal��donie s'est consid��rablement intensifi��e avec des ��pid��mies de plus en fr��quentes, une d��tection du virus en saison fra��che, une co-circulation de plusieurs s��rotypes/g��notypes de dengue ainsi que d'autres arbovirus. En Nouvelle-Cal��donie, le seul vecteur connu de dengue est Aedes aegypti. Consid��rant la dengue comme un probl��me de sant�� publique majeur, la Nouvelle-Cal��donie a rejoint le World Mosquito Prograrn afin de pr��venir les ��pid��mies de dengue par la mise en place d'une strat��gie bas��e sur la bact��rie Wolbachia. Cette strat��gie innovante vise �� diminuer la circulation des arbovirus gr��ce �� des l��chers de moustiques Ae. aegypti porteurs de la bact��rie Wo/bachia, qui emp��chent la r��plication et la transmission du virus de la dengue (DENV). Dans ce contexte, une meilleure compr��hension des m��canismes d'��volution et de transmission virale chez le vecteur, Ae. aegypti,��tait n��cessaire.Ce travail de th��se s'est donc int��ress�� �� l'��tude de la fitness du virus de la dengue chez le vecteur cal��donien Ae. aegypti,au travers de trois objectifs: i) ��valuer la fitness de transmission des quatre s��rotypes de DENV, ii) ��valuer, in vivo, l'impact de la fitness comp��titive de deux g��notypes de DENV-1 sur la transmission virale, et iii) ��valuer l'impact de la bact��rie Wolbachia introduite dans le vecteur cal��donien sur la fitness r��plicative et de transmission du DENV . Ainsi, nous avons pu mettre en ��vidence que le vecteur cal��donien ��tait capable de transmettre les quatre s��rotypes de DENV et qu'il pouvait participer �� la s��lection des souches de DENV participant �� l'��volution du profil ��pid��miologique de la dengue en Nouvelle-Cal��donie. En parall��le, nous avons confmn�� l'impact n��gatif induit par la bact��rie Wolbachia sur cette fitness virale chez notre vecteur. Ces travaux ont notamment permis de d��montrer la complexit�� de la transmission virale chez le vecteur, et de soulever l'int��r��t de conduire des projets de recherche dans un contexte ��pid��miologique pertinent afin de mieux appr��hender la dynamique de la dengue en Nouvelle-Cal��donie, voire dans les autres pays concern��s par cette maladie., In recent years, the circulation of dengue in the Pacifie region and in New Caledonia has intensified considerably with more and more frequent epidemies, detection of the virus in the cool season, co-circulation of severa! dengue serotypes/genotypes as weil as other arboviruses. Aedes aegypti is the on ly known vector for dengue virus (OENV) in New Ca ledonia. Considering dengue fever as a major public health problem, New Caledoniajoined the World Mosquito Program to prevent dengue epidemies by implementing a strategy based on the Wolbachia bacteria. This innovative strategy aims to reduce the circulation of arboviruses by releasing Ae. aegypti mosquitoes infected with Wo/bachia which avoid the replication and transmission of DENV. In this context, a better understanding of the mechanisms of viral evolution and transmission in the vector, Ae. aegypti, was needed. This thesis focused on the study of the DENV fitness in the Caledonian vector Ae. aegypti, through three objectives: i) assessing the transmission fitness of the four DENV serotypes, ii) evaluating the impact of competitive fitness, in vivo, of two DENV-1 genotypes on viral transmission, and iii) measuring the impact of Wo/bachia bacteria introduced into e Caledonian vector on the replicative and transmission fitness ofDENV. Thus, we demonstrated that the Caledonian vector could transmit the four DENV serotypes and that it couId participate in the selection ofDENV strains, participating in the evol ution of the epidemiological profile of dengue fever in New Caledonia. Concurrently, we confinned the negative impact induced by Wo/bachia on this viral fitness in our vector. This work highlighted the complexity of viral transmission in the vector and raises the interest of conducting research projects in a relevant epidemiological context, to better understanding the dengue dynamics in New Caledonia, or even in other countries affected by this disease.

Dengue fever: an emerging infectious disease. Dengue fever is caused by an arbovirus of the family Flaviviridae and the genus Flavivirus, of which there are 4 serotypes (DEN-1, DEN-2, DEN-3, DEN-4). It is transmitted by the bite of a diurnal mosquito of the genus Aedes, mainly A. aegypti and A. albopictus. An increasing cause of acute fever in travellers, it threatens to emerge in temperate regions where competent mosquitoes (Aedes) are established. Dengue fever is characterized by its clinical polymorphism ranging from asymptomatic to severe forms, which are rare in travellers. Its definite diagnosis is based on virological tests selected according to the stage of the disease and the kinetics of the virus. Its treatment is only symptomatic. It is a notifiable disease in mainland France and is subject to a plan to combat its spread and to specific surveillance in the overseas departments. Dengue prevention is based on the application of personal anti-vectorial protection measures among travellers, awareness-raising among health professionals and social mobilization to combat larval gites in endemic regions or regions colonized by Aedes. In France, the tetravalent vaccine Dengvaxia, which is licensed in France, is not recommended for people residing in overseas departments and for travelers to endemic areas.Dengue : une infection émergente. La dengue est due à un arbovirus de la famille des Flaviviridae et du genre Flavivirus, dont il existe quatre sérotypes (DEN-1, DEN-2, DEN-3, DEN-4). Elle est transmise par la piqûre d’un moustique diurne du genre Aedes, essentiellement A. aegypti et A. albopictus. Cause croissante de fièvre aiguë chez les voyageurs, elle menace d’émerger dans les régions tempérées ou des moustiques compétents ( Aedes) sont implantés. La dengue est caractérisée par son polymorphisme clinique allant de formes asymptomatiques à des formes graves, rares chez le voyageur. Son diagnostic de certitude repose sur des examens virologiques choisis en fonction du stade de la maladie et de la cinétique du virus. Son traitement est uniquement symptomatique. Il s’agit d’une maladie à déclaration obligatoire en métropole qui fait l’objet d’un plan de lutte antidissémination et d’une surveillance spécifique dans les départements ultramarins. La prévention de la dengue repose sur l’application des mesures de protection personnelle antivectorielle chez les voyageurs, une sensibilisation des professionnels de santé et une mobilisation sociale pour lutter contre les gîtes larvaires dans les régions endémiques ou colonisées par des Aedes. En France, le vaccin tétravalent Dengvaxia, homologué, n’est cependant pas recommandé chez les personnes qui résident dans les départements ultramarins ni chez les voyageurs se rendant dans des zones endémiques.

Dengue virus (DENV) is part of the Flaviviridae family and has been classify by the Word Health Organization (WHO) as one of the top 10 health concerns. It is the most widespread mosquito-borne human disease. Considering the increasing number of severe dengue, the expansion of the vector territory due to climate change and population movement, it is urgent to find a way to counteract the virus. Indeed, currently there is no treatment available and despite the large number of molecules that proved efficacy in vitro rare are the compounds that have been further evaluated and lead to clinical trials. Development of antiviral is a promising complementary strategy to vaccine production. This review introduces the DENV antivirals and the notions of direct acting antiviral versus host targeted antiviral. It underlines the importance to develop multiple potent antivirals and the relevance to maintain research on this matter.

Dengue virus (DENV) is an arbovirus which is transmitted by Aedes mosquitoes (Aedes aegypti and Aedes albopictus commonly known as tiger mosquitoes). This viral infection is today a major public health problem due to the spread of the vector and for which no therapeutic nor vaccine solution is available. In severe forms, which occur in 1 to 5% of cases (haemorrhagic fevers and life-threatening prognosis), liver damages are observed but their origins are still poorly understood and probably multifactorial. In addition, hepatic steatosis and obesity have been shown to be aggravating factors of the pathology. A growing literature shows that viruses are capable of modulating carbohydrate and lipid metabolisms to promote their replication. In case of DENV infection, it has also been observed that the virus is capable of modulating these cellular metabolic pathways to promote its replication, but the underlying molecular mechanisms are poorly characterized. We have observed that overexpression of DENV NS3 protein increases glycolysis in the hepatocarcinoma cells HuH7. We have also identified interaction of NS3 with the different cellular hexokinases (HK) that control the first step of glycolysis. The expression of HK4, normally and specifically expressed in hepatocytes, restores lipogenesis in HuH7 cells and promotes viral replication. We also identified NS3 as interacting with GKRP, the negative regulator of HK4. Overexpression of GKRP or modulation of its activity on HK4 by metabolic factors such as fructose-6-phosphate, inhibits viral replication. Overall, these observations suggest an interference between NS3 protein and the control of the first step of glycolysis; Le virus de la Dengue (DENV) est un arbovirus transmis par les moustiques du genre Aedes (Ae. aegypti et Ae. albopictus, communément appelé moustique tigre). Le DENV constitue un problème de santé publique majeure du fait de l’expansion du vecteur et pour lequel aucune solution thérapeutique ou vaccinale n’est disponible. Dans les formes sévères de la pathologie (fièvres hémorragiques), on observe une atteinte hépatique dont les causes sont multifactorielles et encore mal connues. Par ailleurs, stéatose hépatique et obésité constituent des facteurs aggravant la pathologie. Une littérature de plus en plus abondante montre que les virus sont capables de moduler le métabolisme glucidique et lipidique pour favoriser leur réplication. Il a également été observé que le DENV est capable de moduler le métabolique cellulaire pour favoriser sa réplication mais les mécanismes moléculaires sous-jacents sont peu caractérisés. Nous avons observé que l’expression de la protéine NS3 du DENV est capable d’induire une augmentation de la glycolyse dans des cellules d’hépatocarcinome HuH7. Nous avons également identifié l’interaction de NS3 avec les différentes hexokinases (HK), premières enzymes de la glycolyse. L’expression d’HK4, spécifique aux hépatocytes, dans des HuH7 restaure la lipogenèse et favorise la réplication virale. Nous avons également identifié l’interaction de NS3 avec GKRP, régulateur négatif d’HK4. La surexpression de GKRP ou la modulation de son activité sur HK4 par des facteurs métaboliques inhibe la réplication virale. L’ensemble des observations suggère une interférence entre la protéine NS3 et la régulation de l’activité HK4 par GKRP, favorisant ainsi l’infection

Severe forms of infection with yellow fever virus (YFV) and dengue virus (DENV) are characterized by liver damage, with more severe symptoms observed during YFV infection. The aim of this thesis is to compare YFV and DENV infections in a model of human hepatocytes derived from stem cells (iHeps) in order to identify factors that could explain their difference in pathogenesis.First, we compared YFV tropism to the four DENV serotypes in 2D iHeps. We observed a low spread of DENV compared to YFV in both iHeps and primary hepatocytes. By using chimeric 17D/DENV strains, we demonstrate that this low propagation is linked to a low DENV entry efficiency in hepatocytes. We also studied infection in iHeps spheroids, metabolically closer to primary cells than 2D iHeps. A productive infection was observed with YFV only. The low accessibility of cells inside the spheroids could explained this result. Second, we studied cellular responses induced following infection by different viruses in 2D iHeps using an RNAseq approach. Preliminary results suggest a link between replication rate and the number of activated genes. The interferon response is detected earlier following YFV infection, but DENV induces a greater number of genes implicated in this pathway. Moreover, DENV-1 and DENV-4 up-regulate some genes involved in antigen presentation such as HLA-E and TAP-2, while YFV down-regulates genes encoding chemokines and adhesion molecules. Preliminary analysis of hepatic metabolism pathways reveals inhibition of the coagulation pathway induced by YFV infection, which is not observed during DENV infection. Similar observations have been described in vivo, at the protein level, confirming the relevance of the iHeps model, Les formes sévères de l’infection par les virus de la fièvre jaune (YFV) et de la dengue (DENV) sont caractérisées par une atteinte du foie, plus sévère lors d’une infection YFV. L’objectif de cette thèse est de comparer les infections de YFV et DENV dans un modèle d’hépatocytes humains dérivés de cellules souches (iHeps) afin d’identifier des facteurs à l’origine de cette différence de pathogenèse. Dans un premier temps, nous avons comparé le tropisme de YFV aux 4 sérotypes de DENV dans notre modèle hépatique établi en monocouche cellulaire. Nous avons observé une faible propagation de DENV dans les iHeps par rapport YFV. Les mêmes observations ont été faites dans des hépatocytes primaires. L’utilisation de souches chimériques 17D/DENV a permis de mettre en évidence que cette faible propagation serait liée à une faible efficacité d’entrée de DENV dans les hépatocytes. Nous avons également étudié l’infection dans des sphéroïdes iHeps, métaboliquement plus actifs que les iHeps 2D. Une infection productive a été observée uniquement avec YFV. Ce résultat pourrait s’expliquer par la faible accessibilité des cellules à l’intérieur des sphéroïdes. Dans un 2ème temps, nous avons étudié les réponses cellulaires induites dans les iHeps 2D après infection par les différents virus en utilisant une approche RNAseq. Les résultats préliminaires suggèrent un lien entre le taux de réplication et le nombre de gènes activés. La réponse interféron est plus précocement détectée dans le cas de YFV, mais l’infection par DENV induit un plus grand nombre de gènes. De plus, DENV-1 et DENV-4 induisent une augmentation d’expression de certains gènes impliqués dans la présentation d’antigène comme HLA-E et TAP-2, alors que YFV diminue l’expression de certains gènes de chimiokines et molécules d’adhésion. L’analyse préliminaire des voies liées au métabolisme hépatique révèle une inhibition de la voie de la coagulation dans le cas de l’infection par YFV, qui n’est pas observée lors de l’infection par DENV. Des observations similaires ont été décrites in vivo, au niveau protéique, confirmant la pertinence du modèle iHeps

Emerging and re-emerging vector-borne viral diseases such as dengue, chikungunya or zika are responsible for many severe epidemics worldwide. Recently, the rapid spread of zika virus (ZIKV) worldwide and the serious neurological complications associated with ZIKV infection have prompted the World Health Organization (WHO) to declare ZIKV a public health emergency. Today, preventive or curative measures against ZIKV infection are almost non-existing. On the other hand, the endemic flora of Reunion Island is known as a rich, renewable and promising source of natural anti-infective products. The registration of 19 medicinal plants from Reunion Island in the French pharmacopoeia highlights the therapeutic potential of natural substances derived from local biodiversity. The work was dedicated to the identification of natural anti-ZIKV substances from a selection of seven medicinal plants from La Reunion registered in the French pharmacopoeia. In a first step, in collaboration with a local start-up (Bourbon Extracts), a solvent-free microwave-assisted extraction was applied to the seven selected plants in order to obtain eco-extracts. In a second step, the screening for antiviral activity, using a ZIKV molecular clone with a reporter gene, allowed the identification of two candidate plants: Aphloia theiformis and Psiloxylon mauritianum. After verifying the absence of cytotoxicity and genotoxicity of the active extracts, the antiviral activity was also demonstrated on other types of extracts using traditional extraction methods (infusion, decoction and maceration). The antiviral activity has been validated on different strains of ZIKV (historical and epidemic) as well as on the four serotypes of the dengue virus. Finally, the mode of action of the two active extracts has been studied. It has been shown that the eco-extracts from A. theiformis and P. mauritianum target the early steps of the viral cycle by inhibiting the attachment of the virus to the host cell. Using electron microscopy, it has been shown that the eco-extract of A. theiformis deforms the viral particle preventing its attachment to the membrane of the host cell. These results demonstrate the importance of medicinal plants from Reunion Island as a source of natural anti-infectious substances., Les maladies virales à transmission vectorielle émergentes et ré-émergentes comme la dengue, le chikungunya ou le zika sont responsables de nombreuses épidémies sévères à travers le monde. Récemment, la propagation rapide et très étendue du virus zika (ZIKV) ainsi que les complications neurologiques graves liées à l’infection par ZIKV ont incité l’Organisation Mondiale de la Santé (OMS) à déclarer le ZIKV comme une urgence de santé publique mondiale. Aujourd’hui, les mesures préventives ou curatives de l’infection par ZIKV sont quasiment inexistantes. D’autre part, la flore endémique de La Réunion est connue comme une source riche, renouvelable et prometteuse en produits naturels anti-infectieux. L’inscription à la pharmacopée française de 19 plantes médicinales réunionnaises souligne le potentiel thérapeutique des substances naturelles issues de la biodiversité locale. Les travaux ont été consacrés à l’identification de substances naturelles anti-ZIKV issues d’une sélection de sept plantes médicinales réunionnaises inscrites à la pharmacopée française. Dans une première étape, une extraction sans solvant assistée par micro-ondes a été appliquée sur les sept plantes médicinales sélectionnées afin d’obtenir des éco-extraits. Dans une deuxième étape, le criblage de l’activité antivirale, en utilisant un clone moléculaire du ZIKV avec un gène rapporteur, a permis d’identifier deux éco-extraits actifs provenant de Aphloia theiformis et de Psiloxylon mauritianum. Après avoir vérifié l’absence de cytotoxicité et de génotoxicité des extraits actifs, l’activité antivirale a été aussi démontrée sur d’autres types d’extraits réalisés via des méthodes d’extractions traditionnelles (infusion, décoction et macération). L’activité antivirale a été validée sur différentes souches de ZIKV (historique et épidémique) ainsi que sur les 4 sérotypes du virus de la dengue. Enfin, le mode d’action des deux extraits actifs a été étudié. Il a été montré que les éco-extraits d’A. theiformis et de P. mauritianum ciblent les phases précoces du cycle viral en inhibant l’attachement du virus à la cellule hôte. À l’aide de la microscopie électronique, il a été montré que l’éco-extrait d’A. theiformis déforme la particule virale empêchant cette dernière de s’attacher à la membrane de la cellule hôte. Ces résultats démontrent l’importance des plantes médicinales réunionnaises comme source de substances naturelles anti-infectieuses.

The flora of New Caledonia, due to its richness and originality, is a potential source of new medicines. Paying particular attention to traditional medicine plants, 48 ​​plants were harvested and 91 crude extracts tested for different biological activities. 10 Plants have provided us with extracts which inhibit the growth of pathogenic microorganisms in man and 15 plants are the source of cytotoxic extracts. These plants could respectively contain new antibiotics and anticancer drugs. 17 plants show strong potential for the research of antiviral compounds, active against the dengue virus. The dengue virus (DV), for which there is no cure today, affects more than 50 million people each year. In order to find inhibitors of this virus, we targeted the DV-NS5 protein, an enzyme essential for replication and specific for DV. The bioguided fractionation of the active extracts of three plants (Myrtopsis corymbosa Guill., Dacrydium balansae Brongn. Et Gris., Carpolepsis laurifolia J. W Dawson) then allowed us to isolate various compounds belonging to the families of alkaloids, coumarins, flavonoids, sterols and terpenes. Among these, flavonoids are the most potent inhibitors of DV-NS5: certain biflavonoids, such as podocarpusflavone A (40), have thus shown a concentration inhibiting 50% of the activity of the viral polymerase (LC50) lower at 1µm, without showing cytotoxicity. The activity of these compounds should now be evaluated against cells infected with the dengue virus.; La flore de Nouvelle-Calédonie, de part sa richesse et son originalité, est une source potentielle de nouveaux médicaments. En portant une attention particulière aux plantes de la médecine traditionnelle, 48 plantes ont été récoltées et 91 extraits bruts testés pour différentes activités biologiques. 10 Plantes nous ont fourni des extraits inhibant la croissance de microorganismes pathogènes de l'homme et 15 plantes sont à l'origine d'extraits cytotoxiques. Ces plantes pourraient respectivement renfermer de nouveaux antibiotiques et anticancéreux. 17 plantes résentent de fortes potentialités pour la recherche de composés antiviraux, actifs contre le virus de la dengue. Le virus de la dengue (DV), contre lequel il n'existe aucun traitement aujourd'hui, touche plus de 50 millions de personnes chaque année. Afin de trouver des inhibiteurs de ce virus nous avons ciblé la protéine DV-NS5, une enzyme essentielle à la réplication et spécifique du DV. Le fractionnement bioguidé des extraits actifs de trois plantes (Myrtopsis corymbosa Guill. , Dacrydium balansae Brongn. Et Gris. , Carpolepsis laurifolia J. W Dawson) nous a alors permis d'isoler divers composés appartenant aux familles des alcaloïdes, coumarines, flavonoïdes, stérols et terpènes. Parmis ceux-ci, les flavonoïdes sont les plus puissants inhibiteurs de la DV-NS5 : certains biflavonoïdes, tels que la podocarpusflavone A (40), ont ainsi montré une concentration inhibant 50% de l'activité de la polymérase virale (CL50) inférieure à 1µm, sans montrer de cytotoxicité. L'activité de ces composés devra désormais être évaluée contre des cellules infectées par le virus de la dengue.

In order to identify new inhibitors of the dengue virus (DENV) NS5 RNA polymerase, a screening was led on 650 new-caledonian plants. Two species, Cryptocarya chartacea Kostermans and Trigonostemon cherrieri Veillon were selected. The EtOAc bark extract of Cryptocarya chartacea (Lauraceae) showed a potent inhibition of the NS5 polymerase activity (99 % at 10 µg/ml). The phytochemical study of the extract led to the isolation of a series of new 6-mono and 6,8-dialkylated flavanones, called chartaceones. Chartaceones are present as racemic mixtures in the plant. Chartaceone A was purified on chiral column leading to the isolation of 4 optically pure diastereoisomers. A configurational study based on the theoretical calculation of circular dichroism spectra allowed the determination of their absolute configuration. Chartaceone are selective inhibitors of the DENV NS5 polymerase. The study of the EtOAc extract from the bark and wood of Trigonostemon cherrieri (Euphorbiaceae) led to the isolation of a series of unusual chlorinated daphnane diterpene orthoesters (DDO) : trigocherrins (non macrocylic) and trigocherriolides (macrocyclic). These compounds inhibit the DENV NS5 polymerase activity and present an antiviral activity on the chikungunya virus in cellulo.; Dans le but de rechercher de nouveaux inhibiteurs de l’ARN polymérase NS5 du virus de la dengue (DENV), un criblage a été mené sur 650 plantes néo-calédoniennes. A la suite de ce criblage, deux espèces (Cryptocarya chartacea Kostermans et Trigonostemon cherrieri Veillon) ont été sélectionnées. L’extrait AcOEt des écorces de Cryptocarya chartacea (Lauraceae) a montré une forte inhibition de la NS5 polymérase (99 % à 10 µg/ml). L’étude phytochimique de l’extrait a mis en évidence une série de nouvelles flavanones 6-mono et 6,8-dialkylées, nommées chartacéones. Celles-ci sont présentes sous forme de mélanges racémiques au sein de C. chartacea. La chartacéone A a été purifiée sur colonne chirale conduisant à l’isolement de quatre diastéréoisomères optiquement purs. Une étude configurationnelle basée sur le calcul théorique de spectres de dichroïsme circulaire a permis la détermination de leur configuration absolue. Les chartacéones inhibent de façon sélective la NS5 polymérase du DENV. L’étude des extraits AcOEt des écorces et du bois de Trigonostemon cherrieri (Euphorbiaceae) a mis en évidence une série de métabolites secondaires originaux de type Diterpènes Daphnane Orthoester (DDO) chlorés : les trigocherrines (non-macrocycliques) et les trigocherriolides (macrocycliques). Ces composés ont montré une inhibition de l’activité enzymatique de la NS5 polymérase du DENV et une activité antivirale sur le virus du chikungunya in cellulo.

Dengue has emerged in Vietnam 50 years ago and since has become endemo-epidemic throughout the whole country. Each year, major epidemics of dengue fever (DF) and dengue hemorrhagic fever (DHF) hit South Vietnam during the rainy season, causing significant morbidity and mortality, especially among young children. The only preventive measure is vector control, but it is often implemented too late or indiscriminately. The aim of this study was to investigate, in the pre-epidemic stage, the existence of significant changes in vector indices, which will predict DF/DHF outbreaks. We conducted a descriptive transversal study, repeated once a month for four months (March to June) in the village of Locthuan (province Ben Tre) in the Mekong's delta. Adult mosquitoes were caught in 30 houses, and larvae were collected in water holding containers of 50 houses. The houses were randomly selected. Vector densities were calculated according to the indices recommended by WHO. Virological analysis was carried out on lots of female Aedes and larvae in order to determine viral infection rates. Catches of adult mosquitoes collected 496 specimens including 329 Aedes, 139 Culex and 28 Anopheles. Aedes aegypti was present in 63% of visited homes that is an average density of 1.8 mosquitoes per house. The increase in imaginal indices during the 4 months was not significant. The survey of breeding sites of Ae. aegypti identified 1292 water containers in which 71,569 larval specimens were collected. The values of house index, container index [CI] and Breteau index [BI] increased each month, the latter from 166 to 442. This increase was significant for CI and BI. Breeding sites were mostly intra-home, mainly consisting of large and small ceramic jars. Larval density of Ae. aegypti in the containers also increased significantly over the 4 months. It was correlated with the lack of cover and predators such as Mesocyclops spp., Micronecta spp. and larvivorous fishes. Cultivation of 15 pools of 10 adult females and 29 pools of larvae (ie 1088 specimens) of Ae. aegypti failed to isolate dengue virus. The high Stegomyia indices measured in this South Vietnamese village and their increase before the rainy season reflect a situation at high risk of epidemics but cannot predict the occurrence of an outbreak in the absence of virus isolation from mosquitoes. They justify conducting an integrated vector control throughout the year.

Considered as major human arbovirosis, dengue occurs in several clinical forms. Some forms can lead to fatal complications such as dengue shock syndrome. This hypovolemic shock cannot be predicted and specific curative treatments are still lacking, and thus management of patients with dengue is difficult. The purpose of this review is to describe state-of-the-art of the knowledge on the pathophysiology of shock syndrome and to highlight the interest of high-content screening methods in translational approaches between research and medicine for investigation of individual response during dengue shock syndrome.

South Western islands of the Indian Ocean are permanently threatened by dengue fever outbreaks. On the Reunion Island, two dengue outbreaks were biologically documented (1977-1978 and 2004). And since July 2004 there has been an inter-epidemic period for the island with sporadic cases and clusters. Between January 1, 2007 and October 5, 2009, the epidemiologic surveillance system detected five confirmed autochthonous cases, five confirmed imported cases (South-East Asia), and 71 probable cases. All the five autochthonous confirmed cases occurred in Saint-Louis during two consecutive clusters. In other South Western islands of the Indian Ocean, several dengue fever outbreaks have been reported. Importation of dengue virus from South-East Asia is a major risk for a new outbreak on the island. The introduction of a new serotype could lead to the emergence of new and severe clinical forms, including dengue hemorrhagic fever.

Aedes albopictus (Tiger mosquito) lives mostly in urban areas. The females lay their eggs in containers close to the stagnant water. Aedes albopictus is spreading worldwide, helped by international transports of tires. In metropolitan France, it has settled down in the French Riviera since 2004, in Corsica since 2006. It is also present in other Mediterranean countries like Italy and Spain. Since Italian outbreak in summer 2007, the risk to acquire an autochthonous chikungunya or dengue diseases in France is high. To limit that risk, the French Health Ministry elaborated in 2006, a national programme. This plan includes preventive actions for patients suspected of chikungunya or dengue after having come back from a tropical area.

International audience; In the 20th century dengue fever became one of the leading causes of morbidity and mortality throughout the tropics. The dengue virus is an arbovirus transmitted by Aedes mosquitoes. There are four distinct serotypes of dengue arbovirus (DENV-1, 2, 3, 4). According to the World Health Organization, a person infected by one of the dengue viruses can develop symptoms ranging from the classical self-limiting form characterized by high temperature, headache, myalgia, and arthralgia to the severe, potentially fatal, form known as dengue shock syndrome. For over 40 years the main explanation for the pathogenesis of dengue has been based on the "antibody-dependent enhancement" (ADE) concept stating that enhancing antibodies acquired during a primary infection increase the number of infected cells, and thus the level of viremia, during secondary infection. However the severity of dengue is not limited to dengue shock syndrome and there are many cases that do not conform to the ADE concept. A meta-analysis could provide crucial information for resolving this controversy and open the way for development of a monovalent vaccine against the dengue virus as for the closely related yellow fever virus.

Dengue (Df) and Chikungunya fever (Cf) arbovirosis are booming in the world, because of the plasticity of their pathogens, mutant RNA viruses making the acquisition of sustainable herd immunity and vaccination difficult in humans, and the plasticity of their vectors, the female mosquitoes of the genus Aedes (Stegomya), capable of adapting to different environments. This review summarizes the viral life cycle and epidemiology of these arboviruses, pathogenesis and pediatric aspects of their clinical forms and the basic principles of their treatment and prevention.

Chikungunya virus (CHIKV) and dengue virus (DENV) are mosquito-borne viruses transmitted by the Aedes genus. Dengue is considered as the most important arbovirus disease throughout the World. Chikungunya, known from epidemics in continental Africa and Asia, has up to now been poorly studied. It has been recently responsible for the severe 2004-2007 epidemic reported in the Indian Ocean (IO), which has caused several serious health and economic problems. This unprecedented epidemic of the IO has shown severe health troubles with morbidity and death associated, which had never been observed before. The two major vectors of those arboviruses in the IO area are Aedes aegypti and Aedes albopictus. The latest is considered as the main vector in most of the islands of the area, especially in Reunion Island. Ae. albopictus showed strong ecological plasticity. Small disposable containers were the principal urban breeding sites, and preferred natural developmental sites were bamboo stumps and rock holes in peri-urban and gully areas. The virus has been isolated from field collected Ae. albopictus females, and in two out of 500 pools of larvae, demonstrating vertical transmission. Experimental works showed that both Ae. albopictus and Ae. aegypti from west IO islands are efficient vectors of dengue and chikungunya viruses. Since 2006 and all along the epidemic of CHIKV, measures for the control of larvae (temephos then Bacillus thuringiensis) and adults (fenitrothion, then deltamethrine) of Ae. albopictus where applied along with individual and collective actions (by the use of repellents, and removal of breeding sites around houses) in Reunion Island. In order to prevent such epidemics, a preventive plan for arboviruses upsurge is ongoing processed. This plan would allow a quicker response to the threat and adapt it according to the virus and its specific vector.

During the first four months of 2003, the survey laboratory of the Federal District (LACEN Laboratory of Virology), Brasília, Brazil, isolated ten strains of dengue virus serotype 3, five of them autochthonous, and the remaining ones from cases imported from Tocantins, Goias and Bahia States. The virus isolations were performed in C6/36 cell culture inoculated with total blood collected between the 1st and the 5th days after the onset of the symptoms. The age of the patients varied from 26 to 59 years old. The strains were typed as DEN-3 by indirect immunofluorescence assay using serotype-specific monoclonal antibodies. Viral RNAs were extracted from total blood using the trizol method. The nested RT-PCR method detected DNA products of 290 bp, confirming the serotype identifications. The introduction of DEN-3 in Brazil and especially in the Federal District represents a serious threat, since most people are susceptible to this serotype and many have already been infected by serotypes DEN-1 or DEN-2, thus increasing the risk of epidemic of more severe forms of the disease. The use of a fast and reliable method for continuous monitoring of the circulation of this serotype is of primary importance for the prevention and control of future epidemics.

Epidemiology of dengue fever is changing dramatically. The worldwide incidence is rising and clinical symptoms are worsening. Reports describing forms associated with haemorrhages or shock syndrome involving both children and adults are increasingly frequent in regions beyond Southeast Asia where the first cases were observed. Many mechanisms could be implicated in these changes, including modifications of the virus, host, vector, or socio-economic factors. The new facilities in the laboratory diagnostic (MAC-ELISA; molecular biology), the commercialization of these assays allow not only assessment of morbidity and mortality in endemic areas and early detection of epidemic outbreaks but also evaluation of socio-economic impact and effectiveness of control measures. Now, the efficiency of the fight must be better, otherwise dengue will grow up during this new century.

International audience; Aedes aegypti is the main vector of dengue viruses. Methods for limiting the spread of dengue outbreaks are currently based on vector control. Estimates of population genetic organization and gene flow with respect to vector capacity have provided great insights into dengue epidemiology. In Vietnam, dengue hemorrhagic fever was detected in the 1950's and becomes today the major problem of public health. Among factors influencing dengue epidemiology, ecological disturbances have a direct impact on vectorial system functioning. Human activities through urbanization creating sanitary conditions are convenient to the vector proliferation and then, to dengue endemisation. To assess the role of the vector in the changing pattern of dengue in South-East Asia, we studied the genetic differentiation and the vector competence towards dengue 2 virus at two scales: at a spatial level (a local scale (i.e., Ho Chi Minh City) and a regional scale (i.e., Cambodia, Thailand and South Vietnam) and at a temporal scale. This study allows to propose a model of Ae. aegypti population functioning according space and time. Dynamics of dengue virus diffusion in relation with the vector, depend on the population genetic composition and its evolution.; Aedes aegypti est le principal vecteur des virus de la dengue dont seule la lutte antivectorielle est capable de limiter l’expansion. Comprendre le mode de fonctionnement des populations du vecteur dans les conditions naturelles est, de ce fait, primordial. En particulier, l’étude de la structure génétique des populations d’Ae. aegypti permettra de mieux définir l’organisation génétique des populations et d’estimer les flux de gènes entre ces populations. Parmi les facteurs susceptibles d’influer sur l’épidémiologie de la maladie, les modifications écologiques ont un impact direct sur le système vectoriel et son fonctionnement. C’est ainsi que les activités humaines jouent un rôle central dans ces changements. L’urbanisation non contrôlée des villes d’Asie du Sud-Est, en générant des conditions sanitaires propices à la pullulation du vecteur, a contribué à l’endémisation de la dengue et à l’émergence puis à la persistance des formes hémorragiques. C’est dans le sud du Vietnam où la dengue constitue un problème majeur de santé publique qu’une étude de la variabilité génétique du vecteur a été initiée. Une analyse de la structure génétique ainsi que celle de la compétence vectorielle des populations ont été menées à une échelle spatiale (de niveau local, la ville de Ho Chi Minh Ville et de niveau régional) et à une échelle temporelle (à différentes périodes de l’année). Ce travail a permis de proposer un modèle de fonctionnement des populations d’Ae. aegypti dans l’espace et dans le temps. Ainsi, la dynamique d’expansion des virus de la dengue en relation étroite avec son vecteur, dépend de la composition génétique des populations de moustiques et de leur évolution dans l’aire de répartition naturelle de l’espèce.

International audience; Dengue is widely distributed in the tropics but epidemic activity was rarely reported in Africa before the 1980's. In the past 15 years, increased epidemic dengue fever has been reported both in East and West Africa, raising concern about the ability of local populations of Aedes aegypti to transmit dengue viruses. Ae. aegypti is present in two forms in Africa: Ae. aegypti aegypti and Ae. aegypti formosus. This latter form, much darker, was not originally a local species but is now colonizing artificial breeding sites within cities. We have been able to demonstrate the oral susceptibility for dengue type 2 virus of Ae. aegypti formosus collected in Franceville, Gabon (Central Africa). However, these mosquitoes sampled exhibited lower infection rates than those of a control colony of Ae. aegypti aegypti originating from French Polynesia.; La dengue sévit dans toute la zone intertropicale mais le continent africain semblait relativement épargné jusqu’au début des années 80. Au cours des 15 dernières années la situation a évolué avec l’apparition d’épisodes épidémiques aussi bien en Afrique de l’Est qu’en Afrique de l’Ouest, soulevant de ce fait le problème de la réceptivité orale pour les virus de la dengue des populations locales d’Ae. aegypti. Ce moustique est présent sous deux formes en Afrique : Ae. aegypti aegypti et Ae. aegypti formosus. Cette dernière forme, non domestique à l’origine, colonise maintenant des gîtes de pontes artificiels dans les villes. Nous avons montré la réceptivité orale pour le virus de la dengue type 2 d’un échantillon d’Ae. aegypti formosus provenant de Franceville (Afrique centrale). Cependant, la réceptivité de cet échantillon était significativement plus faible, à l’issue d’un repas artificiel, que celle d’une souche témoin d’Ae. aegypti aegypti, originaire de Polynésie française.

Two outbreaks of dengue hemorrhagic fever occurred in Guadeloupe (French West Indies) in successive epidemics in 1994 and 1995. The first outbreak was caused by DEN-2 virus and the second by DEN-1.Seven life-threatening infections (WHO grade 3/4) were identified. Three previously healthy adults (including two brothers) died. Autopsy reports (2 patients) disclosed hemorrhagic serous effusions, disseminated intravascular coagulation, and in one case a spontaneous spleen rupture.Dengue fever is an emerging disease. Its severe hemorrhagic form tends to an uprising incidence and can no longer be considered a disease limited to children in Far-Eastern Asia. Fatalities may occur very suddenly and unexpectedly, even in optimal health care settings, in healthy adults living or travelling in endemic areas, notably the Caribbean.

The incidence of dengue increased sharply in Martinique from the end of 1995 into 1996. Virological tests performed jointly on 36 serum samples by the Pasteur Institute in French Guyana and the Center for Disease Control in Puerto Rico led to identification of serogroups 1, 2, and 4 for six dengue virus. Between January 1995 and December 1996, the Departmental Hygiene Laboratory of Martinique carried out screening tests to detect specific IgM by the immunocapture method (MAC ELISA) in patients with suspected dengue. Results were positive in 701 of the 2,143 patients tested (32.7%). Symptoms were studied in 421 of these positive cases. The most frequent presentation was a flu-like syndrome with hyperalgia. Nausea, vomiting, joint pain, and retroocular pain were frequent. At least one clinical sign of coagulation disturbance was noted in 83 patients (19.7%). Dengue hemorrhagic syndrome was diagnosed according to the criteria of the World Health Organization in six patients including one who developed circulatory collapse and died. This fatality was the first to be reported in Martinique. The incidence of typical dengue as well as of the hemorrhagic form is probably underestimated in Martinique because specific serological tests are not routinely requested and application of WHO criteria for diagnosis of hemorrhagic forms is often impractical.

Dengue is a human disease of viral etiology which may be fatal in its hemorragic form. It is widely spread in the tropical areas of the different continents and has been dramatically expanding over the past 30 years. Although an immunological disorder is thought to be involved in dengue physiological symptoms, the pathogenesis of dengue hemorragic fever has not yet been elucidated. Whether the immune response is deleterious or beneficial to the host remains a matter of debate. Other factors, related to virus replication in specific host cells, could also contribute to the severity of the disease. Apoptotic cell death is one of the important consequences of dengue virus infection both in vitro and in vivo. Dengue replication triggers apoptotic signals in neurons and hepatocytes although the original effectors and kinetics differ. Implications of the ongoing apoptotic processes in viral pathogenesis will be further discussed.

Dengue fever is present in tropical and subtropical regions and its geographical extension and the simultaneous increase of its mortality are worrisome. In endemic or epidemic countries, the aim of dengue-specific epidemiological surveillance is to confirm as soon as possible the circulation of a new viral dengue serotype, i.e. the beginning of an epidemic. The efficiency of the control strategy is improved by an earlier epidemic alert. In French Polynesia, dengue-3 virus circulated since 1989 at low level and, in May 1996, a specific epidemiological surveillance was undertaken because of the threat of a dengue-4 epidemic. From each suspected dengue case reported by 18 Polynesian physicians located in the Société Islands, a blood sample was taken for virological assay and clinical data were reported. Between May and November 1996, the virology unit of the Institut Malardé isolated 21 viruses (2 dengue-3 and 19 dengue-2) from 302 suspected cases. The dengue-specific epidemiological surveillance confirmed that dengue-2 virus was circulating and reduced the time of the epidemiological alert by 2 or 3 months compared to previous epidemics. Taking into account the day of illness, a logistic regression undertaken on the clinical data showed that the absence of cough was the only predictive sign of dengue diagnosis. The performance of this dengue-specific epidemiological surveillance system led us to consider its implementation in all concerned countries. A collaboration with international reference laboratories could be a solution for the developing countries.

Yellow fever and dengue are two current viral diseases induced by flaviviruses and usually transmitted by the same mosquito vector, Aedes aegypti. From 1987 to 1991, 18,753 cases of yellow fever, mainly from Africa, have been notified to WHO, leading to 4,522 deaths. On the other hand, WHO estimates that 2.5 billions individuals living in tropical areas are at risk to contract dengue fevers. In fact, 500,000 patients are hospitalized each year for dengue hemorrhagic fever/dengue shock syndrome and 90% of them are children. Nevertheless, the control of these two viral diseases would be reached easily in destroying mechanically the mosquito larval resting places. Although superficially similar, the two entities are in fact quite different. Relatively few is known about the pathogenesis of yellow fever whereas, for dengue fevers, it is difficult to integrate so many results accumulated to explain the occurrence of haemorrhagic phenomena according to the two main theories so far proposed which are not exclusive. The immunological one (S.B. Halstead) tries to explain the pathological events by the effect of anti-dengue enhancing antibodies acquired during a previous exposure to one of the dengue viruses, whereas that of increased virus virulence (L. Rosen) refers to fast passages between individuals during explosive epidemics.

The epidemiology of dengue fever is changing dramatically. The worldwide incidence is rising and clinical symptoms are worsening. Reports describing forms associated with hemorrhage or shock syndrome involving both children and adults are increasingly frequent in regions beyond Southeast Asia where the first cases were observed. Many mechanisms could be implicated in these changes including modifications of the virus, host, vector, or socioeconomic factors. Since no current model allows laboratory analysis of these mechanisms, observation of epidemics is still a major source of data. The findings of well conducted epidemiological studies allow not only assessment of morbidity and mortality in endemic areas and early detection of epidemic outbreaks but also evaluation of socioeconomic impact and effectiveness of control measures. Surveillance techniques must be chosen in function of prevailing local conditions. To ensure reliable results studies should be carried out in collaboration with an international network and a simple and accurate method of disease identification should be used.

The evolution of dengue in French Guiana has the same trend as in most tropical American countries, with emergence of dengue hemorrhagic fever (DHF) and endemicity of the disease. During the 1940's, several dengue-like outbreaks were reported from French Guiana. Then, the only dengue vector, Aedes aegypti, was eradicated between 1950 and 1963, when it started to reinfest the country. By the end of the 1960's, new dengue epidemics with serological confirmation were described. After 1970, the first dengue strains were isolated, firstly dengue-2 strains, then dengue-1 and dengue-4 strains. Between 1970 and 1990, several dengue outbreaks struck French Guiana with 2 to 6 years intervals. In July 1991, started the first DHF outbreak in French Guiana which spread in all the most important towns until October 1992. About 3,000 cases were reported, that is almost 3% of the population, from which about 1/3 had serological confirmation; more than 300 people were hospitalized and some 80 with hemorrhages. Finally 6 deaths were reported, 5 of which had hemorrhages but could not be included into DHF cases according to WHO criteria. The dengue-2 strain responsible for this outbreak is close to the Jamaïcan topotype known to be particularly virulent. Since the beginning of 1993 and actually, dengue is still circulating in French Guiana, but at low level and dengue-1 and dengue-4 strains are occasionally isolated.

Since the 1940s, the dengue epidemics occur more and more often in the Pacific islands with an increased severity. For example, in New Caledonia, outbreaks of dengue-like diseases have been reported since the end of the last century but the first epidemic due to an identified virus occurred in 1942-1943 and was caused by the DEN-1 type. The next, due to the DEN-2 type, was reported thirty years later, in 1972-1973. After that, three outbreaks burst between 1975 and 1990, caused successively by dengue types 1, 4 and 3 but from 1985, human strains of different types were isolated at the same time. The vector control measures, mainly aerial ULV spraying had a variable efficacy. The most important vector was Aedes aegypti but dengue virus strains were also isolated from other mosquito species (Aedes vigilax, Culex annulirostris, C. bitaeniorhynchus, Coquillettidia xanthogaster); their role in the interhuman transmission of dengue was not demonstrated. In New Caledonia, the development of international travels, an extension of suburbs and an increased insecticide resistance of Ae. aegypti are the obvious causes of the problem but an unknown transmission cycle, involving different hosts and vectors may also be suspected.

The situation of dengue in the world can be summarized as follows: 2,5 billions persons at risk, 60 millions cases per year, and 30 000 deaths per year. The four dengue serotypes, DEN-1, DEN-2, DEN-3 and DEN-4 are nearly human-specific. Clinical symptoms of dengue vary from unapparent infection, mild febrile manifestations to hemorrhagic forms with or without shock syndrome. Dengue viruses are transmitted by Aedes mosquitoes, the most important vectors being Aedes aegypti and Aedes albopictus. The epidemiology of the disease can be divided into endemo-epidemic situations occurring in South-East Asia, and epidemic situations found in Pacific islands, Africa and Tropical America. Maintenance of dengue is supported by an increasing demography, uncontrolled urbanization and climatic conditions favorable to the vectors. Spread of dengue is primarily caused by modern transportation means, especially the aircraft. The geographical distribution of dengue is pantropical, except for Madagascar and some african regions. Because no vaccine and no specific treatment actually exist against dengue viruses, mosquito control is the only way to reduce the incidence of dengue around the world. However, the evolution of the dengue situation is not satisfactory.

Prediction and prevention of dengue epidemics are based on informations gathered about the mosquito vector species, the dengue types transmitted, the vertebrate hosts and their environment. Although Aedes aegypti is the most important vector, other Aedes may also propagate the dengue viruses. The populations of vector mosquitoes are evaluated through several indices: Breteau and/or positive house index, number of indoor resting Aedes females, etc.... The four dengue types can replicate in vertebrate hosts beside humans and in other mosquito species than Ae. aegypti. The incidence of dengue on a population is largely variable according to the immunity status, the vector competence and the virus strains. Concomitant infections by two types of dengue virus or by an another pathogen (Alphavirus) have been observed. The environmental factors influencing the dengue ecosystem are mostly climatic (temperature, rainfall, wind) but also anthropic (transportation means, public buildings). Prevention of dengue epidemics must be based on public health education in schools, community participation, epidemiological surveillance linked with good vector control teams. Nevertheless intensive research on dengue and the actions undertaken for the last forty years, dengue remains the first cause of viral morbidity worldwide.