Papers – Dengue & Arbovirosis

2018

An overview of mosquito vectors of Zika virus.
Boyer S, Calvez E, Chouin-Carneiro T, Diallo D, Failloux AB.
Microbes Infect. 2018 Mar 2. pii: S1286-4579(18)30039-X. doi: 10.1016/j.micinf.2018.01.006. 

2017

Dengue-1 virus and vector competence of Aedes aegypti (Diptera: Culicidae) populations from New Caledonia.
Calvez E, Guillaumot L, Girault D, Richard V, O’Connor O, Paoaafaite T, Teurlai M, Pocquet N, Cao-Lormeau VM, Dupont-Rouzeyrol M.
Parasit Vectors. 2017 Aug 9;10(1):381. doi: 10.1186/s13071-017-2319-x.

A neutralization assay for Zika and Dengue viruses using a real-time PCR-based endpoint assessment
Wilson HL, Tran T, Druce J, Dupont-Rouzeyrol M, Catton M.
J Clin Microbiol. 2017 pii: JCM.00673-17. doi: 10.1128/JCM.00673-17.

Socioeconomic and environmental determinants of dengue transmission in an urban setting: an ecological study in Nouméa, New Caledonia.
Zellweger M, Cano J,  Mangeas M, Taglioni F, Mercier A, Despinoy M, Menkès C, Dupont-Rouzeyrol M, Nikolay B and Teurlai M. 
PLoS Negl Trop Dis. 2017 11(4):e0005471.

Zika virus infection and myasthenia gravis: Report of 2 cases.
Molko N, Simon O, Guyon D, Biron A, Dupont-Rouzeyrol M, Gourinat AC.
Neurology. 2017 Feb 10. pii: 10.1212

2016

Vertical Transmission of Dengue Virus in the Peripartum Period and Viral Kinetics in Newborns and Breast Milk: New Data.
Arragain L*, Dupont-Rouzeyrol M*, O’Connor O, Sigur N, Grangeon JP, Huguon E, Dechanet C, Cazorla C, Gourinat AC, Descloux E.
J Pediatric Infect Dis Soc. 2016. pii: piw058.

Global spread of chikungunya virus a lesson for Aedes-transmitted arboviruses.
Duarte dos Santos C, Dupont-Rouzeyrol M, Sam IC, Roques P. CAB
Reviews. 2016 11(18):1-12.

 Zika virus infection as an unexpected finding in a Leptospirosis patient.
Biron A, Cazorla C, Amar J, Pfannstiel A, Dupont-Rouzeyrol M, Goarant C.
JMM Case Rep. 2016 3. doi: 10.1099

Infectious Zika viral particles in breastmilk
Dupont-Rouzeyrol M, Biron A, O’Connor O, Huguon E, Descloux E. .
Lancet. 2016 Mar 12;387(10023):1056.

Differential Susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika Virus
Chouin-Carneiro T, Vega-Rua A, Vazeille M, Yebakima A, Girod R, Goindin D, Dupont-Rouzeyrol M, Lourenço-de-Oliveira R, Failloux AB.
PLoS Negl Trop Dis. 2016 Mar 3;10(3):e0004543.

Whole-Genome Sequencing Analysis from the Chikungunya Virus Caribbean Outbreak Reveals Novel Evolutionary Genomic Elements.
Stapleford KA, Moratorio G, Henningsson R, Chen R, Matheus S, Enfissi A, Weissglas-Volkov D, Isakov O, Blanc H, Mounce BC, Dupont-Rouzeyrol M, Shomron N, Weaver S, Fontes M, Rousset D, Vignuzzi M.  PLoS Negl Trop Dis. 2016 Jan 25;10(1):e0004402.

Genetic Diversity and Phylogeny of Aedes aegypti, the Main Arbovirus Vector in the Pacific.
Calvez E, Guillaumot L, Millet L, Marie J, Bossin H, Rama V, Faamoe A, Kilama S, Teurlai M, Mathieu-Daudé F, Dupont-Rouzeyrol M. 
PLoS Negl Trop Dis. 2016 Jan 22;10(1):e0004374.

2015

Co-infection with Zika and Dengue Viruses in 2 Patients, New Caledonia, 2014.
Dupont-Rouzeyrol M, O’Connor O, Calvez E, Daurès M, John M, Grangeon JP, Gourinat AC.
Emerg Infect Dis. 2015. 21(2):381-2.

Detection of zika virus in urine.
Gourinat AC*, O’Connor O*, Calvez E, Goarant C, Dupont-Rouzeyrol M. 
Emerg Infect Dis. 2015. 21(1):84-6.

2014

Epidemiological and molecular features of dengue virus type-1 in New Caledonia, South Pacific, 2001-2013.
Dupont-Rouzeyrol M, Aubry M, O’Connor O, Roche C, Gourinat AC, Guigon A, Pyke A, Grangeon JP, Nilles E, Chanteau S, Aaskov J, Cao-Lormeau VM. 
Virol J. 2014. 31;11:61.

The emm-cluster typing system for group A Streptococcus identifies epidemiologic similarities across the Pacific region
Baroux N, D’Ortenzio E, Amédéo N, BakerC, Alsuwayyid BA, Dupont-Rouzeyrol M, O’Connor O, Steer A, Smeesters P.
Clin Infect Dis. 2014. 59(7):e84-92.

Isolation and partial characterization of bacteria (Pseudoalteromonas sp.) with potential antibacterial activity from a marine costal environment from New Caledonia.
Dufourcq R, Chalkiadakis E, Fauchon M, Deslandes E, Kerjean V, Chanteau S, Petit E, Guezennec J, Dupont-Rouzeyrol M. 
Lett Appl Microbiol. 2014. 58(2):102-8.

2013

Breast milk as a possible route of vertical transmission of dengue virus?
Barthel A, Gourinat AC, Cazorla C, Joubert C, Dupont-Rouzeyrol M, Descloux E. 
Clin Infect Dis. 2013 Aug;57(3):415-7.

Partial characterization of an exopolysaccharide secreted by a marine bacterium, Vibrio neocaledonicus sp. nov., from New Caledonia.
Chalkiadakis E, Dufourcq R, Schmitt S, Brandily C, Kervarec N, Coatanea D, Amir H, Loubersac L, Chanteau S, Guezennec J, Dupont-Rouzeyrol M, Simon-Colin C.
J Appl Microbiol. 2013 Jun;114(6):1702-12.

2012

Chikungunya virus and the mosquito vector Aedes aegypti in New Caledonia (South Pacific Region).
Dupont-Rouzeyrol M, Caro V, Guillaumot L, Vazeille M, D’Ortenzio E, Thiberge JM, Baroux N, Gourinat AC, Grandadam M, Failloux AB.
Vector Borne Zoonotic Dis. 2012. 12(12):1036-41.

Use of serum and blood samples on filter paper to improve the surveillance of dengue in Pacific Island Countries.
Aubry M, Roche C, Dupont-Rouzeyrol M, Aaskov J, Viallon J, Marfel M, Lalita P, Elbourne-Duituturaga S, Chanteau S, Musso D, Pavlin BI, Harrison D, Kool JL, Cao-Lormeau VM.
J Clin Virol. 2012 Sep;55(1):23-9.

Lower respiratory infections among hospitalized children in New Caledonia: a pilot study for the Pneumonia Etiology Research for Child Health project.
Mermond S, Zurawski V, D’Ortenzio E, Driscoll AJ, DeLuca AN, Deloria-Knoll M, Moïsi JC, Murdoch DR, Missotte I, Besson-Leaud L, Chevalier C, Debarnot V, Feray F, Noireterre S, Duparc B, Fresnais F, O’Connor O, Dupont-Rouzeyrol M, Levine OS. 
Clin Infect Dis. 2012 Apr;54 Suppl 2:S180-9.

2010

Aetiology of community-acquired pneumonia in hospitalized adult patients in New Caledonia.
Mermond S, Berlioz-Arthaud A, Estivals M, Baumann F, Levenes H, Martin PM.
Trop Med Int Health. 2010 Dec;15(12):1517-24.

2008

Real-time PCR detection of gyrA and parC mutations in Streptococcus pneumoniae.
Page S, Vernel-Pauillac F, O’Connor O, Bremont S, Charavay F, Courvalin P, Goarant C, Le Hello S.  Antimicrob Agents Chemother. 2008 Nov;52(11):4155-8.

Leptospira biodiversity

An unexpected Leptospira biodiversity in New Caledonian soils
Principal investigator Cyrille Goarant
Focal point IPNC Roman Thibeaux, Cyrille Goarant
Collaborators at IPNC Marie-Estelle Soupé-Gilbert, Dominique Girault, Emilie Bierque
Other Collaborators Mathieu Picardeau, IPP, Gregorio Iraola and Ignacio Ferres, IP Montevideo
Total budget of project 3000 € Budget devoted to IPNC : NA
Funding IPNC
Timeline Start date: 2016 End date: 2018
Context

Following the demonstration of the environmental survival of pathogenic leptospires at sites of human contamination in New Caledonia, isolation of Leptospira were undertaken.

Infectious strains were not re-isolated from the environment, but a large number of isolates were obtained. Difficulties of identification (MALDI-TOF, 16S rRNA sequencing) led us to suspect novel species and to undertake the sequencing of their complete genome.

Objectives
Perform a genomic study of leptospires isolated from Caledonian soils.
Methods

Isolations were carried out from field soil or water samples. Liquid cultures in EMJH medium were supplemented with a combination of antibiotics described to select growth of leptospires (Chakraborty et al, 2011). The cultures were then spread on EMJH agar medium for isolation.

The isolates were identified with MALDI-TOF and by sequencing their complete genome. Strains of the pathogenic and intermediate clusters are tested in animal models.

Results
More than 100 environmental isolates have been obtained. The first 26 strains for which the complete genome was sequenced showed the presence of 12 novel species: 3 in the pathogen cluster, 5 intermediates and 4 saprophytes.

Strains of the pathogen cluster were unable to cause acute infection in hamsters or renal carriage in mice.

A detailed comparison of core genomes and accessory genomes supports the hypothesis of a polyphyletic group of leptospires with low virulence within the pathogen cluster. This result suggests, in terms of evolution, that virulence was acquired independently in the pathogen and intermediate clusters.

Preliminary results from other isolates suggest other new species.

 

 

Prospects
These 12 novel species should be validly described and named. The new isolates will also be studied. Specific PCR will be designed and tested.
Valorisation 
Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, Ismail N, Mohd Khalid MK, Amran F, Masuzawa T, Nakao R, Amara Khorba A, Bourhy P, Veyrier FJ, Picardeau M, 2019. Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics. PLoS Negl Trop Dis 13: e0007270.

Thibeaux R, Iraola G, Ferrés I, Bierque E, Girault D, Soupé-Gilbert ME, Picardeau M, Goarant C, 2018. Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence. Microbial Genomics 4: 000144.

Thibeaux R, Girault D, Bierque E, Soupé-Gilbert ME, Rettinger A, Douyere A, Meyer M, Iraola G, Picardeau M, Goarant C, 2018. Biodiversity of environmental Leptospira: improving identification and revisiting the diagnosis. Front. Microbiol. 9: 816.

Thibeaux R, Iraola G, Ferrés I, Bierque E, Girault D, Soupé-Gilbert ME, Picardeau M, Goarant C. Of soils, Leptospira and humans. Présentation orale à la conférence de l’ILS, Palmerston North, NZ, November 2017.

 

Biofilm and Leptospira

Project title Leptospira Biofilm: an unexplored reservoir for environmental survival and persistence of infectious bacteria.
Principal investigator Roman Thibeaux
Focal point IPNC Roman Thibeaux, Cyrille Goarant
Collaborators at IPNC Marie-Estelle Soupé-Gilbert, Dominique Girault, Emilie Bierque
Other Collaborators Mathieu Picardeau, IPP, Jean-Marc Ghigo, IPP, Michaël Meyer, UNC
Total budget of project 8000 euros Budget devoted  to IPNCNA
Funding IPNC
Timeline Start: 2018 End :2019
Objectives
The specific objectives of this project are to investigate the bacterial protection provided by the biofilm against both harsh ecological conditions and upon kidney chronic colonization.
Methods

We developed a 96-well method to grow Leptospira biofilm in vitro. After 4 weeks, biofilm formation was quantified using an in-house image analysis workflow based on phase-contrast optical images. To identify leptospiral genes regulating biofilm formation, we quantified impaired or enhanced ability of L. interrogans transposon insertion mutants to produce a biofilm. We then investigated mutants’ infectivity in hamsters, while renal colonization ability was challenged in mice. Protection against environmental stresses (T°, UV light, NaCl) was also assessed using a cell viability Alamar blue assay.

Finally, fluorescently-labelled lectins were used to visualized in situ exo-polysaccharide (EPS) presence and distribution within Leptospira biofilm.

Results

By screening defective mutants, we identified the c-di-GMP signaling pathway as a major regulator of biofilm formation. Indeed, “loss of function” mutants impaired for c-di-GMP synthesis enzymes (GGDEF containing motif) display a decreased ability to produce a biofilm; conversely, “Loss of function” mutants for c-di-GMP recycling enzymes (HD-GYP containing motif) exhibit an enhanced capacity to produce a biofilm. In addition, we show that a defective mutant for an aldo-keto-reductase involved in carbohydrate metabolism exhibits a decreased capacity to produce a biofilm.

We next investigated the ecological relevance of leptospires’ biofilm. Challenge of mutants’ infectivity in hamsters shows that biofilm-deficient mutants are more virulent than mutants displaying an enhanced biofilm production. In a mice model of chronic leptospirosis, urinary excretion load of leptospires was higher for biofilm-deficient mutants as compared to their counterparts. Finally, a protective effect on cell viability was observed for mutants with increased biofilm formation when exposed to high doses of NaCl and UV-c. Further, the increase in viability was strongly correlated to biofilm production in a dose-dependent manner.

Finally, fluorescently-labelled lectins allowed a preliminary characterization of biofilm composition, demonstrating the the presence of α-linked d-glucopyranosyl repeated units as well as x-mannopyranosyl and x-glucopyranosyl residues.

Prospects

This work reveals that, although apparently not required for host infection, biofilm formation improves leptospires’ survival in detrimental environmental-like conditions, thus probably contributing to their persistence in natural habitats.

Ultimately, this work could suggest strategies to impair bacterial biofilm production and organization, thus rendering bacteria accessible to the immune system or harsh ecological conditions. These approaches would hence open new avenues to prevention and treatment of leptospirosis.

Valorisation 
Thibeaux R, Soupé-Gilbert ME, Picardeau M, Goarant C. 2017. Biofilm of pathogenic Leptospira: A compromise between virulence and environmental survival? Présentation orale à la conférence de l’ILS, Palmerston North, NZ, November 2017.

Thibeaux R, Soupé-Gilbert ME, Kainiu M, Girault D, Bierque E, Fernandes J, Bähre H, Douyère A, Eskenazi N, Vinh J, Picardeau M, Goarant C. The Zoonotic Pathogen Leptospira interrogans Mitigates Environmental Stress Through cyclic-di-GMP-controlled Biofilm Production. npj Biofilms and Microbiomes 6(1):24 Texte intégral / Full text

 

MycoplasmaGen-eng

Project title  Mycoplasma genitalium prevalence among men with urethritis
Principal investigator Marion Patoureau and Patrick Blanco (Espas-CMP)
Focal point IPNC A. Tarantola
Collaborators at IPNC A. Tarantola
Other Collaborators J. Colot, M. Biron
Total budget of project 100 000 € Budget devoted to IPNC None
Funding IPNC / Province-Sud
Timeline Start date: 1st Aug 2016 End date : December 2018
Context
The pathogenic role of Mycoplasma genitalium (MG) in non-gonococcal urethritis has recently been recognized. Azithromycin (1 gr. p.o.) is considered as the only effective drug for MG, a pathogen only identified in the early 1980s and the cause of 15-20% of non-gonococcal urethritis and 30% of recurring or persisting urethritis. Rising resistance to azithromycin has been documented. Patients referring for an STI, however, are not screened for MG. Its prevalence as a urethritis-causing pathogen and level of resistance to azithromycin are unknown in New Caledonia. Le rôle pathogène de Mycoplasma genitalium (MG) dans l’urétrite non gonococcique a récemment été reconnu. L’azithromycine (1 gr.p.o.) est considérée comme le seul traitement efficace contre MG, pathogène identifié seulement au début des années 1980 et responsable de 15 à 20% des urétrites non gonococciques et de 30% des urétrites récidivantes ou persistantes. Une résistance croissante à l’azithromycine a été documentée. Les patients référés pour une IST, ne sont cependant pas testés pour MG. Sa prévalence en tant qu’agent pathogène causant l’urétrite et le niveau de résistance à l’azithromycine sont inconnus en Nouvelle-Calédonie.
Objectives
The aim of this study is to estimate the prevalence of Mycoplasma genitalium among men referring for urethritis at the Southern Province walk-in clinic (ESPAS), associated risk factors, resistance profiles if any and the effectiveness of syndromic management. Le but de cette étude est d’estimer la prévalence de Mycoplasma genitalium chez les hommes adressés pour urétrite au centre de soin d’accès libre de la Province du Sud (ESPAS), les facteurs de risque associés, les éventuels profils de résistance et l’efficacité de la prise en charge syndromique.
Methodology
The medical team at ESPAS providing care for men who refer for urethritis complete an anonymized, standardized questionnaire. The data are entered in a computerized datasheet (EpiData), currently being validated by the entry of the first 60 questionnaires. The data will be analyzed by IPNC’s Epidemiology unit. L’équipe médicale de l’ESPAS qui prend en charge les hommes se présentant avec une’urétrite remplit un questionnaire anonymisé et standardisé. Les données sont saisies dans une fiche informatisée (EpiData), en cours de validation par l’entrée des 60 premiers questionnaires. Les données seront analysées par l’unité d’épidémiologie de l’IPNC.
Preliminary results
Validation of the EpiData computerized database Validation de la base de données informatique réalisée sur EpiData
Perspectives
Identifying risk factors associated with urethritis caused by Mycoplasma genitalium to establish a risk profile which will help guide diagnostic management Identifier les facteurs de risque associés avec une uréthrite due à MG pour établir un profile de risque permettant de guider la démarche diagnostique.

REAGIR-eng

Acronym: REAGIR Pyrethroid resistance in Aedes aegypti: evaluation of new insecticides candidates and study of the phenomenon of reversion.
Principal investigator Isabelle DUSFOUR (Institut Pasteur de la Guyane).
Focal point IPNC Nicolas POCQUET
Collaborators at IPNC Laurent GUILLAUMOT, Morgane POL, Sosiasi KILAMA, Marine MINIER.
Other Collaborators Fabrice CHANDRE (IRD), Jean-Philippe DAVID (LECA)
Budget  199 450 € Budget devoted  to IPNC : 54 746 €
Funding The National Agency for Food Safety, Environment and Work (ANSES)
Timeline Start date:
Ending 2014
End date:
End of 2017  (==> extended to 2018)
Context
Aedes aegypti is a mosquito of major medical importance in the intertropical zone, where it transmits arbovirus, especially dengue, chikungunya and Zika viruses. In the absence of any vaccines or effective treatment against these diseases, vector control remains essential. The control of Aedes aegypti is largely based on the use of insecticides, whose effectiveness is now compromised by the development of insecticide resistances. Despite the impact of these resistances on the effectiveness of vector control treatments, there is not much data on the evolution of these resistances in the absence of insecticide pressure.
Objectives
In a context of strong resistance of Ae. aegypti to insecticides in most French overseas territories, this project aims to i) identify new usable active substances and ii) better understand the evolution of resistance to pyrethroids within populations of this species submit to different selection pressures.
Methods
Task 1: populations sampling and assessment of their deltamethrin resistance levels.

Task 2: screening new adulticides by bioassay on laboratories strains of Ae. aegypti.

Task 3: assessment of the possibility to deltamethrin resistance reversion. Four lines are breed in parallel over 10 generations and under different insecticide pressure conditions.

Task 4: evaluation of the counter-selection of deltamethrin resistance for resistant lines subjected to two new insecticidal molecules (identified in task 2).

Task 5: monitoring the evolution of resistance to deltamethrin and the mechanisms involved (i.e. target mutation, expression of detoxification genes) on the experimental lines of tasks 3 and 4.

Results
The populations of French Guyana were found to be much more resistant to deltamethrin than those of New Caledonia (NC). In the absence of insecticide pressure, the deltamethrin resistance level has slightly decreased in NC. On the other hand, the regular introduction of susceptible individuals has had a beneficial effect on reducing the resistance level. The monitoring of the resistance mechanisms shows a weak involvement of the kdr mutations in NC (1011M and 1534C), but a clear correlation between the levels of resistance and expression of some oxidases, in particular CYP9J28. Unfortunately, the majority of candidate insecticides that have proven ineffective in Task 2.
Perspectives
The project aims to provide answers to the problems faced by vector control operators, namely the small number of useful molecules and the development of pyrethroid resistances. At term, this project could provide a strategy to manage Ae. aegypti resistance to this family of insecticides.

DenGen

Acronym: DenGen

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Dengue virus genotype replacements : investigating viral fitness differences driving the evolution of dengue epidemics 
Principal investigator M. Dupont-Rouzeyrol
IPNC main investigator O. O’Connor / M. Dupont-Rouzeyrol
IPNC collaborators N. Pocquet
Other collaborators L. Lambrechts (IP), V. Duong (IPC), P. Dussart (IPC)
Project total budget 50 000 € Budget devoted to IPNC: 21 200 €
Financial supports Actions Concertées Inter Pasteuriennes (ACIP)
Timeline Start date: Jan 2017 End date: December 2018
Context
Phylogenetic analyses have revealed that dengue virus (DENV) evolutionary dynamics are often characterized by the replacement of a DENV genotype by another genotype of the same serotype. Such genotype replacements are epidemiologically significant because they can be associated with changes in disease severity and human immunity. However, the mechanisms underlying DENV genotype turnover in nature remain poorly defined.
Objectives
The specific objectives of this study, led in two different epidemiological contexts: a hyper-endemic area: Cambodia, and an epidemic area: New Caledonia (NC), are: ii) By focusing on vector-virus interactions in vivo, to study the potential role of vector-driven selection in DENV genotype replacements; iii) By focusing on DENV replication kinetics in mammalian cells in vitro, to study the relative ability of DENV genotypes to replicate and produce subgenomic flavivirus (sf) RNAs.
Methods
DENV evolutionary dynamics in NC and Cambodia: About 20 strains per year since 2009 will be selected. E-gene will be sequenced in order to determine the genotype belonging. Based on these results, five representative strains by serotype/genotype will be selected for whole genome sequencing.

DENV competitive fitness in vivo by vector competence assays: Two DENV strains per genotype will be selected for competitive experiment. F1 or F2 generation of Ae. aegypti will be challenged with different ratios of both DENV strains. Infection, dissemination and transmission rates will be measured at day 7 and 14 post-exposure. Virus quantification of both genotype will be performed by RT-qPCR.

DENV replicative fitness in vitro: Replication kinetics of representative DENV strains and production of sfRNA will be observed over 5 days on mammalian cells. RNA quantification will be performed as previously.

Results
All the DENV strains (NC and Cambodia) were obtained after not more than three passages on C6/36 cells. All of them were send to IP for high-throughput sequencing of the whole genome. This one is in progress. Vector competence experiments and in vitro studies are scheduled for the first semester of 2018.
Perspectives
This project will allow us to better understand the evolutionary mechanisms driving DENV genotype shifts typically observed during the course of dengue epidemics.

Acronym: DenGen

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Dengue virus genotype replacements : investigating viral fitness differences driving the evolution of dengue epidemics 
Principal investigator M. Dupont-Rouzeyrol
IPNC main investigator O. O’Connor / M. Dupont-Rouzeyrol
IPNC collaborators N. Pocquet
Other collaborators L. Lambrechts (IP), V. Duong (IPC), P. Dussart (IPC)
Project total budget 50 000 € Budget devoted to IPNC: 21 200 €
Financial supports Actions Concertées Inter Pasteuriennes (ACIP)
Timeline Start date: Jan 2017 End date: December 2018
Context
Phylogenetic analyses have revealed that dengue virus (DENV) evolutionary dynamics are often characterized by the replacement of a DENV genotype by another genotype of the same serotype. Such genotype replacements are epidemiologically significant because they can be associated with changes in disease severity and human immunity. However, the mechanisms underlying DENV genotype turnover in nature remain poorly defined. Des analyses phylogénétiques ont révélé que la dynamique évolutive du virus de la dengue (DENV) est souvent caractérisée par le remplacement d’un génotype du DENV par un autre génotype du même sérotype. De tels remplacements génotypiques sont épidémiologiquement significatifs car ils peuvent être associés à des modifications de la sévérité de la maladie et de l’immunité humaine. Cependant, les mécanismes sous-jacents à ce renouvellement du génotype de DENV dans la nature restent mal définis.
Objectives
The specific objectives of this study, led in two different epidemiological contexts: a hyper-endemic area: Cambodia, and an epidemic area: New Caledonia (NC), are: ii) By focusing on vector-virus interactions in vivo, to study the potential role of vector-driven selection in DENV genotype replacements; iii) By focusing on DENV replication kinetics in mammalian cells in vitro, to study the relative ability of DENV genotypes to replicate and produce subgenomic flavivirus (sf) RNAs. Les objectifs spécifiques de cette étude, menée dans deux contextes épidémiologiques différents: une zone hyper-endémique: Cambodge, et une zone épidémique: Nouvelle-Calédonie (NC), sont : i) étudier in vivo le rôle potentiel de la sélection dirigée par le vecteur dans les remplacements génotypiques du DENV ; ii) étudier in vitro la capacité relative des génotypes du DENV à se répliquer et à produire des ARN subgénomiques du flavivirus.
Methods
DENV evolutionary dynamics in NC and Cambodia: About 20 strains per year since 2009 will be selected. E-gene will be sequenced in order to determine the genotype belonging. Based on these results, five representative strains by serotype/genotype will be selected for whole genome sequencing.

DENV competitive fitness in vivo by vector competence assays: Two DENV strains per genotype will be selected for competitive experiment. F1 or F2 generation of Ae. aegypti will be challenged with different ratios of both DENV strains. Infection, dissemination and transmission rates will be measured at day 7 and 14 post-exposure. Virus quantification of both genotype will be performed by RT-qPCR.

DENV replicative fitness in vitro: Replication kinetics of representative DENV strains and production of sfRNA will be observed over 5 days on mammalian cells. RNA quantification will be performed as previously.

Dynamique évolutive du DENV en NC et au Cambodge: environ 20 souches par an depuis 2009 seront sélectionnées. Le gène E sera séquencé afin de déterminer l’appartenance du génotype. Sur la base de ces résultats, cinq souches représentatives par sérotype / génotype seront sélectionnées pour le séquençage du génome entier.

Fitness compétitive du DENV in vivo par des analyses de compétence vectorielle: Deux souches de DENV par génotype seront sélectionnées pour l’expérience de compétition. Une génération F1 ou F2 d’Ae. aegypti sera mise en contact avec différents ratios des deux souches du DENV. Les taux d’infection, de dissémination et de transmission seront mesurés aux jours 7 et 14 après l’exposition. La quantification virale des deux génotypes sera effectuée par RT-qPCR.

Fitness réplicatif du DENV in vitro: La cinétique de réplication de souches représentatives du DENV et la production de sfRNA seront observées pendant 5 jours sur des cellules de mammifères. La quantification de l’ARN sera effectuée comme précédemment.

Results
All the DENV strains (NC and Cambodia) were obtained after not more than three passages on C6/36 cells. All of them were send to IP for high-throughput sequencing of the whole genome. This one is in progress. Vector competence experiments and in vitro studies are scheduled for the first semester of 2018. Toutes les souches du DENV (NC et Cambodge) ont été obtenues après moins de trois passages sur des cellules C6/36. Elles ont toutes été envoyées à l’IP pour le séquençage à haut débit du génome complet. Celui-ci est en cours de réalisation.
Perspectives
This project will allow us to better understand the evolutionary mechanisms driving DENV genotype shifts typically observed during the course of dengue epidemics. Ce projet nous permettra de mieux comprendre les mécanismes évolutifs qui sous-tendent les remplacements de génotypes du DENV typiquement observés au cours des épidémies de dengue.