Posted on by Marc Jouan

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

logo intnetwork

 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.