|Acronym: DenGen||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|
|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.|
|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.|
|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.
|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.|
|This project will allow us to better understand the evolutionary mechanisms driving DENV genotype shifts typically observed during the course of dengue epidemics.
|Acronym: DenVect||Vector competence of Aedes aegypti for Dengue viruses in New Caledonia|
|Principal investigator:||O. O’Connor|
|IPNC main investigator:||O. O’Connor, M. Dupont-Rouzeyrol|
|IPNC collaborators:||C. Inizan, N. Pocquet|
|Budget||8380 €||Budget devoted to IPNC:|
|Timeline||Start date: Aug.2016||End date: July 2017|
|Occurence of dengue outbreaks needs three key actors: the pathogen (i.e. the virus), the vector (Aedes sp.) and the human host. In New Caledonia, Ae. aegypti is the major (the only proven to date) vector of arboviruses. Since World War II, NC was regularly affected by dengue outbreaks with a cycling circulation of the four DENV serotypes. However, by the beginning of the year 2000, this pattern has changed with the persistence of DENV-1. These observations suggest that our local vector is competent for DENV. However, its capability to be infected by DENV, allowing the replication and transmission of the virus to human host, has not been studied yet.|
|We propose to investigate the ability of New Caledonian Ae. aegypti population to transmit different DENV serotypes, in link with the epidemiological profile observed.|
Selection of DENV strains from viro-bank and virus stock production: one representative DENV strain of each serotype/genotype will be selected in IPNC viro-bank depending on the year of epidemic circulation. The selected strains will be amplified on C6/36 cells with no more than three passages. Quantification of viruses obtained will be performed by immunofluorescent focus assay on C6/36 cells.
Phenotypic characterization of representative strains in the vector: An Ae. aegypti F1 generation will be used for vector competence studies carrying out on each representative DENV strains. Two independent experiments will be performed, each with the four DENV serotype strains. Infection, dissemination and transmission rates will be measured at day 7 and 14 post-exposure. Samples analysis will be performed as previously described.
|DENV-1 to -4 strains, phylogenetically characterized, were obtained on C6/36 cells. The two independent vector competence experiments were conducted with all DENV strains. Preliminary results obtained from the first experiment show that New Caledonian Ae. aegypti are capable to transmit DENV. Transmission of DENV-1 (genotype I “Asian”) and DENV-4 is observed as early as 7 days post-exposure with rates of 20% and 33% respectively. At 14 days post-exposure, the transmission efficiency is varying from 12.5% to 22% for all the DENV strains. Analysis of the second experiment is in progress.|
|In New Caledonia, the context is particularly favourable to study the dynamic of DENV transmission, as Ae. aegypti is the only proven arboviruses vector and the epidemiology of dengue is well-kown. Thus, this project will allow us to have a global vision of the Ae. aegypti competence at a given time, which is an important point to better understand the transmission and dynamics of DENV in New Caledonia.|
|2017 Dengue outbreak, Investigations of severe cases : Dengue Severity|
|Principal investigator||M. Dupont-Rouzeyrol|
|IPNC main investigator||M. Dupont-Rouzeyrol|
|IPNC collaborators||A. Tarantola, C. Inizan, M. Minier, O. O’Connor|
|Other collaborators||E. Simon-Lorière, A. Sakuntabhai (IPP), E. Descloux, M. Sérié, M.-A. Goujart,, A.-C. Gourinat (CHT), C. Forfait, A. Pfannstiel (DASS-NC).|
|Budget||Budget devoted to IPNC|
|Financial supports||IPNC, IPP, CHT, NC|
|Timeline||Start date: Sept.2017||End date : Sept. 2018|
|With 4,401 dengue cases (2,548 biologically confirmed), 579 hospitalized cases and 11 deaths between Jan 1st and Oct 8th 2017, the 2016-2017 dengue epidemic in New Caledonia (NC) was sizeable. More importantly, it was associated with a higher percentage of hospital admissions and severe clinical presentations (liver injury, ophthalmic complications…). The higher occurrence of deaths and heretofore rare hepatic forms observed during this epidemic may be linked with specific characteristics of viruses, patients, or host-pathogen response.|
|To determine whether severe presentations of dengue infections were associated with specific characteristics such as virus genotype and hepatic tropism, patient comorbidities or earlier infection by dengue or Zika viruses.|
|Patients already included in the ArboVirtuess study will be asked to continue their participation and contribute an additional blood sample.
– Selection of 50 sera from 2017 and previous years available for analysis: 1/3 severe forms versus 2/3 non-severe
– Phylodynamic studies based on whole-genome sequencing. Search for variations in viral quasi-species.
– Comparative analysis of the hepatic tropism of viral strains representative of 2016-2017 epidemic versus previous years
– Sera characterization: history of previous arbovirus infections, impact of pre-existing humoral immunity on DENV replication in human monocytes (ADE)
|Ethical approval for patient recall have been obtained and sera have been selected and collected. Viral RNAs have been extracted from the sera and whole-genome sequencing is in progress. In parallel, viruses from this year’s epidemic are currently being isolated on mosquito C6/36 cells and will be titrated. Experimental conditions for human hepatic cell line infection as well as for ADE have been set up and are currently being validated. Characterization of viruses’ hepatic tropism and sera inhibition/enhancement capacity will soon be initiated.|
|First, this study may shed light on a potential evolution of viruses involved in severe cases compared to non-severe dengue cases, in link with a modification in virus hepatic tropism. Second, this project will provide an unprecedented characterization of the impact of anti-Zika antibodies on DENV infection. Overall, this study is expected to further the understanding of the unusual severity of DENV epidemic in 2017. Such documentation of the influence of a pre-existing anti-Zika humoral immunity on dengue severity would be useful for territories displaying an epidemiological situation similar to NC, thus suggesting a new indicator to estimate the risk of progression towards severe dengue.|
|Acronym: VIP||Are dengue Defective Interfering Particles truly interfering?|
|Principal investigator||C. Inizan|
|IPNC focal point||C. Inizan / M. Dupont-Rouzeyrol|
|IPNC collaborators||O. O’Connor|
|Project total budget||8 275 €||Budget devoted to IPNC||NA|
|Timeline||Start: March 2017||End: Feb. 2018|
|Polymerase errors occurring upon viral replication might give rise to incomplete genomes that are nevertheless encapsidated in Defective Viral Particles (DVP). Replicated and maintained upon co-infection with functional viruses, such DVPs might affect the physiopathology of the infection: inhibition of viral replication, modulation of the host immune response as well as contribution to infection persistence. In the case of dengue virus (DENV), DVPs have been detected in both humans and Aedes mosquitoes. They might represent a natural regulator of viremia in patients, thus impacting infection evolution and resolution.|
|The current study aims at evaluating DVP role in viremia regulation in patients. This project includes (i) the characterization and quantification of DVPs in the sera of dengue patients, in link with viremia and, (ii) the measure of DVP impact on DENV replication in vitro.|
|Molecular characterization of DVPs in the sera of dengue patients from New Caledonia:
– Evaluation of patients viremia through in vitro infections and real-time RT-PCT
– Quantification and analysis of DVPs subgenomic RNA profiles in patients’ sera through real-time RT-PCR
– Sequencing of DVPd subgenomic RNAs in patients sera
Measure of the modulation of DENV replication in vitro by DVPs:
– Production and purification of DVPs through in vitro infections and ultracentrifugation
– Quantification of DVP impact on DENV replication in human cells in vitro through real-time RT-PCR
|Viral infectious titers of 50 sera of patients from New Caledonia infected by DENV-1 genotype I have been evaluated through in vitro infections. In parallel, viral RNAs have been extracted from those sera. Molecular analyses are being set up and will soon enable the quantification and sequencing of DVPs subgenomic RNAs.|
|Through the implementation of DVPs detection and purification tools, the current project opens new avenues for a broader study on DVPs role in DENV natural cycle, aiming at measuring their impact on dengue physiopathology and transmission. This study of the impact of DVPs on viremia might suggest their quantification in patients’ sera as a prognostic tool for infection severity.|
|Acronym : R-Zero||Risk factors for Zika virus Emergence and Recurrent Outbreaks within the Pacific area.|
|Principal investigator||VM. Cao-Lormeau (ILM)|
|IPNC main investigator||M. Dupont-Rouzeyrol|
|IPNC collaborators||D. Girault, A. Tarantola|
|Other collaborators||Ministère de la santé de Fidji, Samoa et Yap, Institut Pasteur, London School of Hygiene and Tropical Medicine, Queensland University of Technology|
|Project total budget||32 000€
|Budget devoted to l’IPNC||3 000 €|
|Financial supports||Pacific Funds (phase 1/3)|
|Timeline||Start date: Jul 2017||End date: July 2018|
|In 2014, Zika (ZIKV) emerged in the Pacific region and spread in the world. In early 2016, WHO declared the ZIKV outbreak an international public health emergency in connection with the severe neurological consequences observed following ZIKV infection. Since then, the number of countries reporting a Zika epidemic has steadily increased, illustrating that ZIKV’s geographic expansion is still ongoing, even in the region. In addition, the reappearance of ZIKV in the Federated States of Micronesia, Kosrea, 8 years after the Yap epidemic, illustrates the risk of outbreak’s recurrence in previously exposed areas.|
|The overall objective of this project is to identify the risk factors for the emergence and recurrence of Zika epidemics in the Pacific, with a particular focus on anti-Zika community immunity.|
|Characterization of the memory immune response against ZIKV – persistence and possible interference with dengue
Community mmunity against ZIKV – Status and Evolution in 4 Pacific sites
Epidemic dynamics of Zika – risk factors for epidemic emergence and recurrence
|This project is expected to gather crucial information on the persistence of protective immunity against Zika virus in infected patients. It should also be used to assess the level of exposure and immunity of Pacific populations to the Zika virus.|
|Communication : M. Dupont-Rouzeyrol, R-Zero Workshop, Tahiti, 2017 (Oral)|
|Acronym : ZikAlliance||A global Alliance for Zika Virus control and prevention.|
|Principal investigator||X. de Lamballerie (Inserm)|
|IPNC main investigator||M. Dupont-Rouzeyrol|
|IPNC collaborators||N. Pocquet, O. O’Connor, M. Minier|
|Other collaborators||AB. Failloux (IP), ZikAlliance consortium, task 6.2|
|Project total budget||11.9M€||Budget devoted to IPNC:||30 000 €|
|Timeline||Start date: Jan. 2017||End date: December 2019|
|In 2014, Zika (ZIKV) emerged in the Pacific region and spread in the world. In early 2016, WHO declared the ZIKV outbreak an international public health emergency in connection with the severe neurological consequences observed following ZIKV infection. The ZikAlliance project is a large project that aims to study ZIKV and its consequences at different scales and across different environments. IPNC is a partner of this project and is involved in the WP6: Vectors and Vector control and more particularly the vector competence part. The objectives presented here correspond only to the task in which IPNC is a partner.|
|Evaluate the vector competence of several mosquitoes species and populations from different environments (American Asia, Africa…) against several ZIKV lineage|
|Homogenization of protocols.
Generation of F1 populations of Aedes aegypti and Culex quinquefasciatus from New Caledonia.
Production of Zika virus stocks, different lineages.
Vector competence experiments: measurement of infection, dissemination and transmission parameters.
Analysis of the results at the global scale.
|Two populations of Ae. aegypti (Nouméa and Koné) were generated. The ZIKV African lineage and Asian / American lineage were received. Experimental infections are scheduled for the first half of 2018.|
|Mieux connaître la capacité de transmission des différents vecteurs pour les différentes lignées est nécessaire à l’évaluation du risque et à la mise en place de méthodes de lutte et de prévention plus ciblées et plus performantes.|
|Publications : Dupont-Rouzeyrol et al, In press, Calvez et al, submitted ; Boyer et al, submitted; Simon et al, submitted ; Calvez et al, in prep.|