Vincent Raquin

Rift Valley Fever Virus (RVFV) is an arbovirus responsible for significant mortality and morbidity in both humans and animals. Classified by the WHO as a priority pathogen, RVFV is at risk of worldwide emergence notably due to a large vector species spectrum. As other arboviruses, RVFV vector competence (VC) is driven by genetic and environmental factors. Understand how these factors shape RVFV transmission by mosquitoes is therefore critical to mitigate Rift Valley fever emergence and spread. Conversely to dengue, chikungunya or Zika viruses, little is known on the impact of major VC drivers such as mosquitoes species or virus dose for RVFV. In addition, some potential VC drivers have been overlooked for most arboviruses, such as the cellular origin of viruses used in VC assays. Finally, intra-vector infection dynamics (DIV), represented by the distribution of extrinsic incubation period (EIP) within the mosquito population, remains a black box for many vector-arbovirus pairs. Here, we solved some of these gaps by feeding Aedes aegypti and Culex quinquefasciatus mosquitoes with the RVFV ZH548 reference strain prior to detect, at various time post RVFV exposure, infectious virus in mosquito tissues representative of infection, dissemination and transmission stages. These data were then used to model RVFV DIV for both mosquito species. Major VC variations were observed according to mosquito species, virus dose and cell line used for virus stock production, in a stage-specific manner. Furthermore, key differences were unveiled between the DIV of Ae. aegypti and Cx . quinquefasciatus mosquitoes. This study highlights the pattern of RVFV dynamics in mosquitoes according to major VC drivers, and provides a reference data set upon standardized experimental VC assay in two major RVFV vector genera ( Aedes and Culex ). Altogether, this work opens new avenues towards the understanding RVFV-mosquito interactions, and how they can modulate RVFV emergence and spread. AUTHOR SUMMARY Rift valley fever virus (RVFV) is an understudied but threatening mosquito-borne pathogen, both from human and animal health perspective. The limited number of studies, notably on mosquito capacity to transmit this virus (vector competence), prevents us to fully understand RVFV epidemiology and mitigate emergence. Here, I explored key aspects of RVFV transmission by exposing mosquitoes to a reference virus strain through an artificial blood meal. Results show that the mosquito species, the dose of virus in the blood meal or the way the virus is prepared prior to add it into the blood meal impact vector competence. Each factor above displays stage-specific impact on RVFV-mosquito interaction, modulating either initial mosquito infection, virus dissemination within the mosquito cavity or virus spread in vector’s saliva. Finally, modeling tools applied to vector competence data showed that each mosquito species present a specific balance between susceptibility (% of mosquitoes infected) and time to infectiousness (median number of days to switch from infected to disseminated stage). Altogether, this work uncovers key features of mosquito vector competence for RVFV, explains previous literature discrepancies and highlights the benefit of combining experimental and modeling work to link vector competence and epidemiological studies.

On assiste actuellement à l'émergence et la ré-émergence mondiale d'arboviroses comme le chikungunya, la dengue ou la fièvre de la vallée du Rift. Ces maladies, responsables d'environ 30 000 décès par an, sont dues à des virus principalement transmis à l'homme par des moustiques vecteurs. En l'absence de vaccins efficaces et face aux limites de l'utilisation d'insecticides, nocifs pour les écosystèmes et entrainant des résistances chez les vecteurs, il est nécessaire de développer des moyens alternatifs de lutte. La découverte récente du potentiel antiviral de certaines bactéries symbiotiques du moustique, comme le genre Wolbachia, représente un outil de lutte biologique prometteur face aux arboviroses. Ce projet de thèse porte sur la relation tripartite moustique-bactéries endosymbiotiques-arbovirus, en prenant pour modèle le moustique-tigre, Aedes albopictus. Cette espèce originaire d'Asie envahit progressivement l'Europe. Elle transmet notamment le virus de la dengue et du chikungunya, et est naturellement infectée par Wolbachia. Les résultats obtenus ont permis d'observer un phénotype antiviral chez les moustiques infectés par Wolbachia, contrairement aux moustiques aposymbiotiques. L'utilisation d'une méthode de transcriptomique haut-débit (RNAseq) a permis de déterminer certains mécanismes cellulaires et moléculaires majeurs du moustique spécifiquement impliqués dans l'interaction avec les arbovirus, Wolbachia, et les deux partenaires simultanément. Le développement d'une lignée cellulaire d'Ae. albopictus stablement infectée par Wolbachia a permis de mettre en évidence le rôle central de l'autophagie dans l'interaction Wolbachia-arbovirus chez Ae. Albopictus

Arboviruses represent a significant threat to the health of humans, animals, and plants worldwide. Mechanistic modeling has proven useful for elucidating the transmission, anticipating the spread, and predicting the response of arboviruses to control measures. However, most models approximate the intra-vector infection dynamic (IVD), which occurs during the extrinsic incubation period (EIP), by a single stage with an average duration. At the end of this stage, all exposed vectors are expected to become infectious. Strong assumption is hidden behind this: that the EIP is exponentially distributed in the vector population. To assess the validity of this assumption, we developed a stochastic compartmental model that represents successive IVD stages, associated with the crossing or not of the three within-mosquito barriers (infection, dissemination, and transmission). We calibrated the model using an ABC-SMC (Approximate Bayesian Computation - Sequential Monte Carlo) method, which includes model selection. We searched for literature data on experimental infections of Aedes mosquitoes infected by dengue, chikungunya, or Zika viruses. We demonstrated the large discrepancy between the exponential hypothesis and observed EIP distributions for dengue and Zika viruses, and identified more relevant EIP distributions. This work provides a generic modeling framework that can be applied to other arboviruses for which similar data are available. Our model also can be linked to population-scale models to aid future arbovirus control efforts.

Impact statement: The Asian tiger mosquito, Aedes albopictus, is an invasive species that spreads diseases like chikungunya and has caused outbreaks worldwide. Studies show that mosquito‐associated microbes can affect disease transmission. One of those microbes, the parasite Ascogregarina taiwanensis, is common in native and settled mosquito populations (>3 years after introduction) but rare in recently introduced ones. We found that this parasite slows down the spread of the chikungunya virus within the mosquito and decreases its transmission rate by half. Unparasitized mosquitoes spread the virus more easily, suggesting that changes in mosquito‐associated microbes could impact disease outbreaks and public health.

While locally-acquired dengue virus (DENV) human infections occur in mainland France since 2010, data to identify the mosquito species involved and to trace the virus are frequently lacking. Supported by a local network gathering public health agencies and research laboratories, we analysed, in late summer 2023, mosquitoes from privately-owned traps within a French urban neighbourhood affected by a dengue cluster. The cluster, in Auvergne-Rhône-Alpes, comprised three cases, including two autochthonous ones. Upon return from a recent visit to the French Caribbean Islands, the third case had consulted healthcare because of dengue-compatible symptoms, but dengue had not been recognised. For the two autochthonous cases, DENV-specific antibodies in serum or a positive quantitative PCR for DENV confirmed DENV infection. The third case had anti-flavivirus IgMs. No DENV genetic sequences were obtained from affected individuals but Aedes albopictus mosquitoes trapped less than 200 m from the autochthonous cases’ residence contained DENV. Genetic data from the mosquito-derived DENV linked the cluster to the 2023–2024 dengue outbreak in the French Caribbean Islands. This study highlights the importance of raising mosquito-borne disease awareness among healthcare professionals. It demonstrates Ae. albopictus as a DENV vector in mainland France and the value of private mosquito traps for entomo−virological surveillance.

Vector competence assays allow to measure, in the laboratory, the ability of a mosquito to get infected and then retransmit an arbovirus while mimicking natural vector infection route. Aedes aegypti is a major vector of arboviruses worldwide and thus a reference species used in vector competence assays. Rift Valley fever virus (RVFV) is a major public health threat, mostly in Africa, that infects humans and animals through the bite of mosquito vectors. Here, we describe vector competence assay of Aedes aegypti mosquitoes for RVFV, from mosquito exposure to the virus through an infectious artificial blood meal to the measurement of virus prevalence in the mosquito’s body, head, and saliva.

Along their life cycle, mosquitoes colonize different ecological niches with various microorganisms and diet sources that likely modulate their performance (i.e. a set of mosquito fitness-related traits). However, which ecological parameters and how their variations modulate mosquito performance is not completely understood. In this study, we used Ae. albopictus surface-sterilized eggs re-associated or not to conventional bacterial microbiota upon a range of diet concentrations and addressed the impact of microbial inoculum and diet concentration variation on several mosquito performance traits. Results showed that mosquito juvenile survival depends on the interaction between bacterial inoculum load and diet concentration in the breeding water. Exposure to bacteria in rearing water shorten larval development time although it impacted larvae survival in an inoculum and diet concentration-dependent manner. Bacterial composition of larval rearing water was mainly structured by the bacterial inoculum concentration, with some Operational Taxonomic Units abundances correlating with larval traits. Ae. albopictus survival, development and bacterial community patterns upon gradients of diet and bacterial inoculum illustrated the complex impact of diet-microbiota interaction on mosquito performance. These findings argue the importance of deciphering host-microbe-environment interactions and open promising perspectives to improve Ae. albopictus control measures in the field. IMPORTANCE: Microbiota is increasingly recognized as a driving force of metazoan biology, impacting diverse traits including nutrition, behaviour or reproduction. The microbial impact on host nutrition is among the most studied host-microbe interactions although it remains poorly understood in arthropod vectors like mosquitoes. Here, we manipulated mosquito microbiota using gnotobiology to decipher the impact of bacteria and diet on the Asian tiger mosquito, Ae. albopictus. These results are key to understand the link between diet and bacteria concentration on juvenile mosquitoes as well as carry-over effects in adults. They unveil some specific aspects of mosquito-bacteria interactions while opening interesting avenues for vector management of this vector of arboviruses.

Arbovirus emergence and epidemic potential, as approximated by the vectorial capacity formula, depends on several host and vector parameters including vector intrinsic ability to transmit the pathogen. Such ability, called vector competence is influenced by biotic (e.g. virus and vector genotype) and abiotic (e.g. temperature). Vector competence is often evaluated as a qualitative phenotype although it is a multistep, time-dependent, quantitative phenotype. Combination of experimental and modelling approaches can i) capture intra-vector dynamics of arboviral infection and ii) use data to estimate arbovirus epidemic potential. Here, we measured individual Aedes albopictus (Lyon, France) mosquitoes infection, dissemination, and transmission rate upon oral exposure to chikungunya virus (CHIKV, La Reunion Island isolate) at eleven time-points from day 2 to day 20 post-exposure (dpe) for a range of CHIKV infectious doses spanning human viremia. Statistical modelisation indicates an explosive CHIKV intra-vector dynamics mainly due to an absence of dissemination barrier with 100% of the infected mosquitoes ultimately exhibiting a disseminated infection regardless of the viral dose. Transmission rate data revealed a time and dose-dependent but overall weak transmission barrier with individuals transmitting as soon as 2 dpe and >50% infectious mosquitoes at 6 dpe for the highest dose. Epidemiological simulations conducted with an Agent-Based Model based on experimental intra-vector dynamics data showed that even at low mosquito biting rates, CHIKV triggers explosive outbreaks. Together, this reveals the high epidemic potential of this CHIKV isolate with this French metropolitan population of Aedes albopictus.

Mosquitoes are considered one of the most important threats worldwide due to their ability to vector pathogens. They are responsible for the transmission of major pathogens such as malaria, dengue, zika, or chikungunya. Due to the lack of treatments or prophylaxis against many of the transmitted pathogens and an increasing prevalence of mosquito resistance to insecticides and drugs available, alternative strategies are now being explored. Some of these involve the use of microorganisms as promising agent to limit the fitness of mosquitoes, attract or repel them, and decrease the replication and transmission of pathogenic agents. In recent years, the importance of microorganisms colonizing the habitat of mosquitoes has particularly been investigated since they appeared to play major roles in their development and diseases transmission. In this issue, we will synthesize researches investigating how microorganisms present within water habitats may influence breeding site selection and oviposition strategies of gravid mosquito females. We will also highlight the impact of such microbes on the fate of females’ progeny during their immature stages with a specific focus on egg hatching, development rate, and larvae or pupae survival.

Diseases caused by mosquito-borne viruses have been on the rise for the last decades, despite the implementation of vector control methods primarily based on insecticides. An alternative control method currently in development is the use of lab-engineered mosquitoes that are incapable to carry viruses. This has stimulated efforts to identify optimal target genes that are naturally involved in mosquito antiviral defenses or required for viral replication. Although several antiviral immune pathways such as RNA interference (RNAi) have been previously characterized in mosquitoes, the genes that prevent or promote early viral replication in the midgut remain elusive. Here, we investigated the role of a member of the Tudor protein family, Tudor-SN, upon dengue virus infection in the mosquito Aedes aegypti . Tudor-SN expression was upregulated early after dengue virus infection and was subsequently positively correlated with viral loads in the midgut. Using RNAi-mediated knockdown, we showed that the loss of Tudor-SN reduced dengue virus replication in the Ae. aegypti derived cell line Aag2 and in the midgut of Ae. aegypti females in vivo . Using immunofluorescence assays, we found that Tudor-SN localizes to the nucleolus in both Ae. aegypti and Aedes albopictus cells. Finally, we used a reporter assay to demonstrate that Tudor-SN was not required for RNAi function in vivo . Collectively, these results define a novel proviral role for Tudor-SN upon early dengue virus infection of the Ae. aegypti midgut.