Influence of biotic factors on Rift Valley Fever virus infection dynamics in mosquitoes

Influence of biotic factors on Rift Valley Fever virus infection dynamics in mosquitoes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of biotic factors on Rift Valley Fever virus infection dynamics in mosquitoes Daphné Baudon, Barbara Viginier, Léa Loisel, Marie-Pierre Confort, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9196651/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 its large vector species spectrum. Understanding how genetic and environmental (a)biotic factors shape RVFV transmission by mosquitoes is therefore critical to prevent Rift Valley fever emergence and spread. Studies often focused on main vector competence (VC) drivers such as mosquitoes species or virus dose, for arboviruses currently considered as major human threats worldwide like dengue, chikungunya or Zika viruses. Other potential VC drivers have been overlooked, like the cellular origin of viruses used in VC assays, while some mosquito-borne viruses remain understudied including RVFV. In addition, intra-vector infection dynamics (IVD), represented by the extrinsic incubation period (EIP) distribution 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 reference RVFV ZH548 strain prior to measure viral infection, dissemination and transmission in individual mosquitoes and estimate RVFV IVD. Major VC variations were observed according to mosquito, virus dose and cell line used for virus stock production together with key differences in IVD between Ae. aegypti and Cx . quinquefasciatus . This study provides a reference data set of mosquito VC for RVFV, for a range of host-like virus doses and stocks, including the EIP range for the two major RVFV vector genera ( Aedes and Culex ). Altogether, this work opens new avenues towards the understanding RVFV-mosquito interactions, and how they impact RVFV emergence and spread. Rift Valley fever virus arbovirus mosquito vector competence DIV Aedes aegypti Culex quinquefasciatus Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Arthropod-borne viruses (arboviruses) are transmitted to vertebrate hosts by arthropod vector during blood-feeding. Arboviruses infections account for 28.8% of (re)-emerging viral infectious diseases 1 , causing hundreds of millions cases and representing a major public health burden 2 . Arbovirus transmission is driven by population and individual-level interactions between host, vector and pathogen under the influence of environmental parameters. Establish how variations in these factors impact disease transmission, within areas at risk or endemic for an arbovirus, is a fundamental basis of risk mitigation strategies. For example, the vector species, the virus strain and the viral dose to which the vector is exposed during a blood-meal can modulate vector competence (VC, i.e. , vector’s ability to become infected and then transmit a pathogen), which in turn can influence disease dynamics. However, this has not been thoroughly investigated for several arbovirus-vector pairs such as RVFV in mosquitoes. Moreover, the experimental design (virus production method, vector blood feeding strategy and the number of time points post virus exposure) can hinder our ability to capture the full dynamics of VC notably its time component, the extrinsic incubation period (EIP). Rift Valley Fever virus (RVFV) was first reported in 1930 in Kenya, in the Rift Valley 3 and is now widespread in Africa 4 – 8 where it has caused hundreds of human deaths, significant livestock losses and major socio-economic impacts over the past 100 years 9 . Over the past 40 years, RVFV spread to the Indian ocean in 1990 10 then in Arabia Peninsula in 2000 11,12 pushing the World Health Organisation to consider RVFV as a priority pathogen with worldwide spread potential 13 . RVFV transmission potential is partly linked to its wide host range, the virus being found a variety of wild animals as well as in livestock, including bovine and ovine. RVFV is also transmitted by large spectrum of vectors mainly mosquitoes of Aedes and Culex genera, with at least 30 mosquito species being identified as potential vectors 14 , 15 . Some species have been both found positive for RVFV in the field and competent under laboratory conditions including Aedes aegypti , Aedes mcintoshi , Aedes vexans , Culex pipiens and Culex quinquefasciatus 15 . Therefore, they represent relevant targets for RVFV-mosquito studies. As observed for other arboviruses, mosquito VC for RVFV depends on both mosquito and virus genotypes despite this is based on a limited number of studies 16 – 18 . Host viremia level is another key determinant of VC as suggested by direct mosquito feeding on viremic hamsters, although these studies did not allowed to estimate dose-VC relationship 20 . The duration and amplitude of RVFV viremia in vertebrate hosts varies across species, such as sheep were it ranges from 5 to 7 log 10 TCID 50 /mL with a peak at day 3 post-infection 19 , 21 . In this context, the use of an artificial blood feeding allows to avoid animals and provides a standardized and reproducible 22 assay. In addition, artificial blood feeding allows to spike the blood meal 22 , 23 with a viral stock of controlled genetic composition and cellular origin, via the use of reverse genetics systems and selected amplification cell lines respectively. To our knowledge, the impact of the cell line used for RVFV stock production on VC has not been addressed, and no proper RVFV dose-response experiment was performed upon a range of host viremia-like doses. Recently a stochastic compartmental model of intra-vector infection dynamics (IVD) was developed which, when combined with a tailored experimental design, improves our estimation of VC and EIP distribution 24 . While the IVD is critical to characterize the EIP distribution, it has not yet been characterized for RVFV. This study aims to estimate VC of a reference RVFV strain (ZH548) produced by reverse genetics from various cell lines for two mosquito laboratory colonies (Ae. aegypti and Cx. quinquefasciatus ) exposed to host-representative virus doses. Using this approach, a unique dataset was generated demonstrating that the mosquito, the viral dose and the cell line used for viral stocks production influence VC for RVFV although at different steps of the mosquito infection cycle. Estimating RVFV IDV showed that the time required to switch from an infected to a disseminated stage is mosquito-species dependent, providing critical insights for RVFV transmission potential modelling. This work represents an important step towards the understanding of RVFV-mosquito interactions, that will help to improve prediction and mitigation of RVFV (re)emergence worldwide. MATERIALS AND METHODS Cell culture Two widely used cell lines for RVFV production, namely VeroE6 produced in monkey kidney (kindly provided by ANSES) and C6/36 produced in Ae. albopictus mosquito larvae (European Collection of Authenticated Cell Cultures, reference 89051705), were used. In addition, MDBK, IDO5 and CPT-Tert 25 , originating from, bovine kidney, sheep derma and sheep choroid plexus respectively, were also used for RVFV stock production. Mammalian cells were maintained at 37°C, 5% of CO 2 in 75cm 2 flasks and passaged once a week (1:10 dilution). VeroE6, MDBK and IDO5 were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma) supplemented with 4% (v:v) foetal bovine serum (FBS, Dominique Dutscher), 1% (v:v) penicillin-streptomycin solution (PS, Gicbo) (Penicillin 5U/mL and Streptomycin 5µg/mL) and 1% (v:v) sodium pyruvate. CPT-Tert were cultured in Iscove Modified Dubelcco Madie (Gibco) with 10% FBS and 1% PS. Mosquito C6/36 cells were maintained at 28°C, without CO 2 in 25cm 2 flasks and passaged weekly (1:10 dilution) in Leibovitz’s L-15 Medium (L-15, Gibco) supplemented with 10% of FBS, 10% Tryptose Phosphate Broth (Sigma) and 1% PS. Virus stock production and titration The complete genomic sequence of RVFV ZH548 strain (isolated from a symptomatic child in Egypt in 1977 and belonging to the genetic lineage A 26 ) was used to generate a reverse genetic clone (ZH548-RG). Briefly, ZH548 segment L, M and S (consensus sequences were deposited in GenBank with accession numbers OR805805, OR805806, OR805807) were cloned into a pTVT7 plasmid downstream of a T7 promoter. The three plasmids were transfected in hamster kidney cells BSR T7/5 CL21 (kindly provided by Prof. Alain Kohl, Liverpool 27 ) to produce an initial virus stock (P0) that was amplified once in VeroE6 cells (P1) and titrated By plaque forming assay using VeroE6 cells (titers are expressed as plaque forming units per mL, PFU/mL) 28 . The P1 stock was then inoculated at a multiplicity of infection of 0.001 on the five cell lines described above to obtain the working virus stock (P2). Briefly, cells were seeded the day prior infection at 5 and 6 log 10 cell per 25-cm 2 flask for mammalian and insect cells, respectively. After 3h, virus suspension was removed and 5 mL of fresh media were added per flask. Infection was stopped upon observation of cytopathic effects (CPE) for mammalian cells (typically between 2- and 4-days post-inoculation) or at 3 days for C6/36 cells (which do not display CPE). Cell supernatants were harvested, centrifugated 4 min at 1000 G to remove cell debris and then stored at -80°C as aliquots. A mock (flask without virus) was also prepared concomitantly as control. Two independent P2 stocks were produced, namely P2-stock1 and P2-stock2. The P2-stock1 and P2-stock2 infectious titers were measured by fluorescent focus assay (FFA) on C6/36 cells 29 . The day prior infection, 5.5 log 10 cells/well were seeded into 96-well plates (TPP). After removing cell culture media, 50 µL of a serial dilution (1:10) of each P2 stock were added per well. Plates were incubated for 1 h at 28°C then a 150 µL overlay of a 1:1 (v:v) mix of cell culture media and 3.2% carboxymethyl cellulose solution (Sigma) were added per well. The plates were incubated for 3 days at 28°C, then the cells were fixed using 150 µL/well of a 4% paraformaldehyde solution in 1X Dulbecco’s phosphate buffered saline (DPBS, Gibco) for 20 min at room temperature. After 3 washes in 1X DPBS, cells were permeabilized for 30 min at room temperature using 40 µL/well of 0.3% (v:v) Triton X-100 (Sigma) in 1X DPBS + 1% Bovine Serum Albumin (BSA, Sigma). After 3 washes in 1X DPBS, 40 µL/well of RVFV anti-N antibody (produced in rabbit), kindly given by Dr Benjamin Brennan (MRC—University of Glasgow Centre for Virus Research—Glasgow) 30 and diluted 1:1000 in 1X DPBS + 1% BSA were added in each well as a primary antibody. Cells were incubated for 1h30 at 37°C, rinsed 3 times in 100 µL of 1X DPBS and then incubated for 1 h at 37°C with 40 µL of Alexa546-conjugated anti-rabbit secondary antibody (produced in goat) (ThermoFisher) diluted at 1:2000 in 1X DPBS + 1% BSA. Cells were rinsed 3 times in 1X DPBS, once with tap water and stored at 4°C protected from light. Plates were analysed within a week under a Colibri 7 fluorescence microscope (Zeiss) under the 10X objective. The number of fluorescent foci was counted and the infectious titer was expressed as fluorescent focus forming unit per mL (FFU/mL). The absence of viral signal was confirmed in wells inoculated with cell culture media only (negative controls). For the P2-stock1, FFA titration was performed in parallel on C6/36 and VeroE6 cells using the same protocol. Mosquito colonies maintenance Laboratory colonies of Ae. aegypti PAEA 31 (Anna-Bella Failloux, Institut Pasteur Paris) and Cx. quinquefasciatus SLAB (Grégory L’Ambert and Marie-Laure Setier, EID Méditerranée) were used. Colonies were maintained at IVPC laboratory biosafety level 2 insectary in mass rearing under standard conditions (26°C, 70% relative humidity, 12:12 hours light:dark cycles) using Hemotek membrane feeding system and human blood (from multiple anonymous donors collected by EFS AURA under the CODECOH agreement DC-2019-3507). The Cx. quinquefasciatus colony was maintained continuously (no egg drying) while Ae. aegypti egg papers were stored at 26°C, 70% relative humidity for up to 4 months. Larvae were maintained in 23 x 34 x 7 cm plastic trays (Gilac) with dechlorinated tap water supplemented with a 3:1 (w:w) mix of tropical fish food (Tetramin) and yeast powder (Biover). Adults were maintained in 30 x 30 x 30 cm cages (Bioquip) at 26°C, 70% relative humidity, 12:12 h light:dark cycle with permanent access to 10% (m:v) sugar solution. Mosquito artificial infectious blood meal The detailed protocol used here for mosquito artificial blood meal is available regarding Ae. aegypti 23 , although slight adjustments were done regarding Cx. quinquefasciatus . Fifteen days prior the infectious blood feeding, Ae. aegypti eggs were placed in dechlorinated tap water in a vacuum chamber (-400 mm Hg) at 26°C for ~ 12h while Cx. quinquefasciatus were offered a blood meal in order to obtain first-instar larvae the day before the infectious blood meal. Synchronized first-instar larvae from both species were then transferred in plastic trays at a density of 200 larvae/tray, with 1.5 L of dechlorinated tap water. Optimal larval development was obtained by adding 0.1 g of food in each tray every two days. The day prior infectious blood meal, 4 to 7-day old females were placed in 136 x 81 mm plastic feeding boxes (Corning-Gosselin) with 45 to 60 females per box and 1 male. Females were transfered in the level 3 biosafety facility (SFR AniRA, Lyon Gerland) at 26°C, 80% of humidity, 12:12h light:dark cycle with no access to sugar solution. A human erythrocyte suspension was prepared the day prior the infectious blood meal. Briefly, human total blood was diluted v:v in PBS, centrifugated 15 min at 1500 G and the supernatant was removed, this operation being performed three times in total. The resulting washed erythrocytes suspension was mixed 2:1 (v:v) with viral suspension, and supplemented with 2% 0.5 M ATP as a phagostimulant (Sigma). Feeders (Hemotek) were covered with pig small intestine and filled with 3 mL of infectious blood mixture. Females were allowed to feed for 20 minutes at 26°C in presence of smelly socks and regular short C0 2 puffs (one every 5 min) to promote feeding. Bloodmeal aliquots were taken before (T0) and immediately after (T + 1 hour) the feeding and stored at -80°C for further titration. Mosquitoes were anesthetized on ice ( Ae. aegypti ) or with C0 2 ( Cx. quinquefasciatus ) and only fully engorged females were transferred in 1-pint cardboard containers (~ 15–25 females/container) with permanent access to cotton soaked with 10% sucrose solution. Cardboard containers were placed in 18 x 18 x 18 inches cages (BioQuip) and incubated at 26°C, 70% humidity, 12:12 h light:dark cycle. Vector competence assays Individual mosquitoes and, depending on the experiment, saliva were collected at several days post RVFV exposure (dpe) according to the experimental design. For saliva collection, females were anesthetized as described above then legs and wings were removed. Individuals were placed on a plastic plate maintained by double-sided adhesive tape. The proboscis was inserted in a trimmed 10 µL filtered tip containing 10 µL of FBS and held by modelling clay 32 . 2 µL of 1% pilocarpine hydrochloride solution (Sigma) supplemented with 0.1% Tween-20 (Sigma) in water were added on the thorax of each mosquito to promote salivation. Mosquitoes were allowed to salivate at 26°C, 70% relative humidity during 45 minutes. The content of the tip was then expelled in tube filled with 100 µL of DMEM media (Gibco) supplement with 4% FBS and antibiotics (Amphotericin B 2.5µg/mL, Nystatin 10 000 U/mL, Gentamicin, 50µg/mL, Penicillin 5U/mL and Streptomycin 5µg/mL (Gibco)). Of note, saliva samples were deposited immediately (no freezing) on VeroE6 cells in order to maximise the detection of RVFV infectious particles. Following saliva collection, or immediately upon harvest when salivation was not performed, mosquitoes were stored at -80°C. On the day of infectious titration assay, mosquitoes were thawed and individually processed. Mosquito heads and bodies were separated using a pin holder with a 0.15 mm minutien pins (FST). Samples were transferred in individual grinding tubes containing 400 µL of DMEM supplemented with FBS and antibiotics (see above) and one 3-mm diameter tungsten bead (Qiagen). Samples were grinded using a TissueLyzer II (Qiagen) for 2 rounds of 1 min at 30 Hz then centrifugated 5 min at 1000 G and stored at -80°C. RVFV detection was performed by FFA titration assays on VeroE6, as previously described, using 50 µL or 100 µL of raw sample for bodies and heads, respectively, to determine the infectious status ( i.e. , positive or negative for infectious RVFV). Positive (RVFV viral stock) and negative (grinding media) controls were added on each titration plate. This approach allows to estimate vector competence based on three parameters: the infection rate (number of RVFV-positive mosquito bodies / number engorged mosquitoes), the dissemination rate (number of RVFV-positive heads / number of RVFV-positive bodies) and the transmission rate (number of RVFV-positive saliva / number of RVFV-positive heads). Statistical analysis Infectious titer (expressed as log 10 (FFU/mL)) was considered as continuous response variable and the cell line used for virus production and for virus titration were considered as discrete explanatory variables. Mosquito infection, dissemination and transmission rate were considered as binary response variables (infected or not) while time post virus exposure (dpe) and virus dose in the blood meal were treated as continuous explanatory variables. When several independent experimental replicates were performed, the impact of the experiment effect (discrete variable) on the response variable of interest was tested. If no significant experiment effect was found, data from the different experimental replicates were grouped for downstream analysis. A full factorial linear mixed model (gaussian error and an identity link function) was used to estimate the effect of the cell line used for virus stock production and virus titration on the viral infectious titer while controlling for random variation among the different independent stocks produced. Post-hoc Tukey-HSD pairwise tests were applied to perform pairwise comparisons between infectious titres between cell lines. A full factorial generalized linear mixed model (GLMM), with a binomial error and logit link function was used to determine the effect of cell line and dpe and their interaction on mosquito infection rate by RVFV, while controlling for random variation among experimental replicates. Despite no significant experiment effect, GLMM could not be ran for other analyses. Therefore, full factorial generalized linear model (GLM) were used to test for the impact of cell line, mosquito species, virus dose and dpe on mosquito infection rate, dissemination rate, transmission rate or transmission efficiency (number of RVFV-positive bodies / number engorged mosquitoes) depending on the experimental design, excluding dpe for transmission rate when transmission was measured only at a single dpe. All the statistical analyses and figures were produced under R environment (RStudio, POSIT) with the main packages ggplot2,Tidyverse 33 , plyr 34 , emmeans 35 and multcomp . Raw data and scripts used in this study are available (). Modelling RVFV intra-vector dynamics RVFV intra-vector dynamics was described using a discrete-time stochastic compartmental model (IVD model) with daily time steps, as previously developed and detailed 24 . This model represents the same processes observed in the vector competence experiments. It includes one compartment for each IVD stage 24 . Stage E represents exposed mosquitoes, when the virus is in the digestive tract following a blood meal. Stage I corresponds to infected mosquitoes, when the virus is present in the midgut cells. Stage D denotes disseminated mosquitoes, when the virus has spread to the circulatory system. Finally, stage T refers to transmitter or infectious mosquitoes, marked by the presence of the virus in the saliva. To account for the non-systematic barrier crossing 36 , three parameters, \(\:{\gamma\:}_{I},{\gamma\:}_{D}\:and\:{\gamma\:}_{T}\) were introduced to represent the proportions of mosquitoes for which the infection, dissemination, and transmission barriers were crossed, respectively. To model the distributions of durations in the I and D stages, these compartments were subdivided into several sub-compartments representing possible residence times. Mosquitoes were randomly distributed among these sub-compartments according to either an exponential ( \(\:\lambda\:\) ) or a beta distribution (parameters \(\:\alpha\:\) and \(\:\beta\:\) ), allowing for flexible shapes of time distributions. The model was implemented in R. Each stochastic model replicate, defined by the parameter set (γ_I,γ_D,γ_T,(α_I,β_I) or λ_I,(α_D,β_D) or λ_D ), represented a single vector competence experiment and produced outputs comparable to those obtained in laboratory experiments, namely the numbers of mosquitoes in the infected, disseminated, and transmitter stages at each observed day post exposure (DPE). Model calibration Model parameters were inferred using an Approximate Bayesian Computation (ABC) approach with a Sequential Monte Carlo (SMC) sampler 37 and model selection 38 , following the framework described in 24 and implemented in the R package BRREWABC. This process included the selection of the best model among four options: IbetaDbeta, IbetaDexpo, IexpoDbeta, and IexpoDexpo. These models differ based on the distribution of duration used—either beta or exponential—for the infected and disseminated stages, as indicated by the first and second parts of their names, respectively. Depending on the selected model, five or seven parameters were inferred: the three barrier-crossing proportions ( \(\:{\gamma\:}_{I},{\gamma\:}_{D},{\gamma\:}_{T}\) ) and the distribution parameters for stages I (( \(\:{\alpha\:}_{I},{\beta\:}_{I}\) ) or \(\:{\:\lambda\:}_{I}\) ) and D (( \(\:{\alpha\:}_{D},{\beta\:}_{D}\) ) or \(\:{\lambda\:}_{D}\) ). The ranges of variation of each of inferred parameters and their justifications are presented in supplementary Table SX. Calibration was performed using aggregated data from two experimental replicates with Ae. aegypti (PAEA) and Cx. quinquefasciatus (SLAB) females exposed to 6.7 log 10 FFU/mL of RVFV in the blood meal and harvested at 3, 7, 10, 14, and 21 dpe. For each simulated parameter set (particle), distances between observed and simulated values were computed as the sum of squared errors across the four IVD stages. Particles were accepted when all distances were below stage-specific thresholds. Thresholds were adaptively updated at each iteration based on the 90th percentile of accepted distances, and 800 particles were retained per round. This inference yielded parameter sets describing intra-vector viral dynamics in PAEA and SLAB mosquitoes, allowing comparison of residence-time distributions in I and D and their variation between the two species. RESULTS RVFV intra-vector dynamics varies according to the mosquito species Two independent experiments were performed in which both Ae. aegypti and Cx. quinquefasciatus females were simultaneously exposed to a blood meal spiked with ~ 6.7 log 10 FFU/mL of a RVFV ZH548-RG strain. The virus dose was stable in the blood during the time course of the infectious meal (Figure S1A). At the selected days post RVFV exposure (dpe), individual mosquitoes were tested for the presence of infectious virus in the body, head and saliva as a proxy of infection, dissemination and transmission rates, respectively (values shown as % with the associated 95% confidence interval). The infection rate ranged from 21% [12–34] to 57% [42–71] with no significant impact of the experiment (LR, Chisq = 0.85, Df = 1, P experiment = 0.35). RVFV infection in mosquitoes did not significantly vary according to dpe (LR, Chisq = 0.89, Df = 1, P dpe = 0.34), the mosquito species (LR, Chisq = 0.006, Df = 1, P species = 0.93) or their interaction (LR, Chisq = 0.13, Df = 1, P species*dpe = 0.71) (Fig. 1 A). These results were consistent across the two independent experiments (Figure S1B). Among RVFV infected mosquitoes, virus dissemination ranged from 0% [0–21] to 70% [49–85] in Ae. aegypti and from 30% [14–53] to 42% [21–66] in Cx. quinquefasciatus (Fig. 1 B) without significant experiment effect (LR, Chisq = 0.94, Df = 1, P experiment = 0.32) (Figure S1C). RVFV dissemination depended on the interaction between dpe and mosquito species (LR, Chisq = 6.54, Df = 1, P species*dpe = 0.01) (Fig. 1 B). Of note, this interaction was significant for one of the two experimental replicates (LR, Chisq = 12.15, Df = 1, P species*dpe = 0.0004, experiment 1) but not in the other (LR, Chisq = 0.07, Df = 1, P species*dpe = 0.79, experiment 2), where only the dpe significantly impacted dissemination (LR, Chisq = 5.05, Df = 1, P dpe = 0.02, experiment 2) (Figure S1C). Overall, RVFV dissemination increases over time, although this increase depends on the mosquito species, indicating a vector-specific dynamic of RVFV dissemination (Fig. 1 B). The proportion of saliva with infectious RVFV virus ranged from 11% [0.005–0.49] to 83% [36–99] (Fig. 1 C), the large confidence intervals being driven by the small sample size, due to previous infection and dissemination barriers, in absence of experiment effect (LR, Chisq = 0.04, Df = 1, P experiment = 0.82) (Fig. 1 A-B). The impact of the interaction between species and dpe on transmission rate was bearly significant (LR, Chisq = 3.87, Df = 1, P species*dpe = 0.049) when analysing the two experiment together. However, this interaction was not significant for either of the experiments separately (LR, Chisq = 0.007, Df = 1, P species*dpe = 0.93 for experiment 1 ; Chisq = 3.57, Df = 1, P species*dpe = 0.058 for experiment 2), while the effect of dpe remained significant in both experiments (LR, Chisq = 5.66, Df = 1, P dpe = 0.01 for experiment 1 ; Chisq = 3.93, Df = 1, P dpe = 0.04 for experiment 2) in absence of species effect (LR, Chisq = 0.76, Df = 1, P species = 0.39 for experiment 1 ; Chisq = 2.46, Df = 1, P species = 0.11 for experiment 2) (Figure S1D). Altogether, these data show that RVFV transmission is likely driven by the dpe, with the proportion of infectious mosquitoes increasing over time (Fig. 1 C). The transmission efficiency (TE, proportion of mosquitoes with RVFV in the saliva out of the total number of engorged specimens), which encompasses previous infection, dissemination and transmission steps to provide a proxy of RVFV transmissibility by the vector, was calculated (Fig. 1 D). Results indicated that TE depends on the interaction between dpe and mosquito species (LR, Chisq = 11.1, Df = 1, P species*dpe = 0.0008), a result that is consistent across experiments (Figure S1E). This result indicates that, beyond experimental variations, Ae. aegypti and Cx. quinquefasciatus are stably infected by RVFV in the midgut, allowing for increasing viral dissemination over time, which peaks after 14 dpe. However, vector competence vary according to the species, with Ae. aegypti being overall more competent than Cx. quinquefasciatus for RVFV ZH548-RG strain, likely due to a higher dissemination rate. To further explore RVFV intra-vector dynamics (IVD) in mosquitoes, including the EIP, our recently developed modelling tool 24 was used to estimate RVFV IVD in Ae. aegypti and Cx. quinquefasciatus using the vector competence data presented above (Fig. 1 A-C). Model selection step showed that the estimation of infection (I) and dissemination (D) step durations best fit a beta (I) / exponential (D) distribution for Ae. aegypti (Fig. 2 A). In contrast, for Cx. quinquefasciatus , both exponential (I) / exponential (D) and, to a lesser extent, exponential (I) / beta (D) fitted the observed distribution. Using the best fit for both species, the model suggested that the majority of Ae. aegypti mosquitoes spent more time in (I) with a peak at ~ 10 days compared to Cx. quinquefasciatus for which most of the individuals left (I) in fewer than 5 days (Fig. 2 B). Once in (D), the time spent at this stage was comparable for both species (Fig. 2 C). Overall, vector competence and IVD estimation for the two mosquito species showed that Ae. aegypti has a higher dissemination rate but takes longer to disseminate RVFV compared to Cx. quinquefasciatus . Cell line used for RVFV stock production impacts the early stages of mosquito infection The Ae. aegypti mosquito population was chosen for subsequent experiments, as it displayed a higher vector competence overall for our RVFV ZH548-RG strain compared to Cx. quinquefasciatus (Fig. 1 ). We now aim to investigate the impact of the virus-producing cell line on vector competence. Stocks of ZH548-RG strain were produced using five different cell lines (VeroE6 and C6/36, MDBK, CPT-Tert and IDO5) reaching 7–8 log 10 FFU/mL, regardless of whether VeroE6 or C6/36 was used for virus titration (Figure S2A). No significant difference in viral titer was observed between the two cell lines used for it. Viral RNA:infectivity ratio was calculated and shown that C6/36-derived RVFV stocks present a lower ratio compared to mammalian cells (Figure S2B). Virus doses in the blood meal did not vary according to time or producer cell line, remaining at ~ 6.5 log 10 FFU/mL in both experiments (Figure S2C). The infection rate ranged from 5% [1–13] to 34% [22–48] with no impact of the dpe while controlling for experiment effect (GLMM) (Wald χ 2 , Chisq = 1.10, Df = 1, P dpe = 0.29) (Fig. 3 A and S2 D-E). However, the producer cell line used significantly influenced mosquito infection rate (Wald χ 2 , Chisq = 18.5, Df = 4, P stock = 0.0009). As mosquito infection rate did not depend on the dpe, data from 7, 14 and 21 dpe were grouped to interrogate the impact of the RVFV producer cell line on the infection rate while controlling for experiment effect (Fig. 3 D). Post-hoc tests showed that RVFV produced in C6/36 had a higher infection rate compared to MDBK ( P C6/36 v MDBK = 0.046, Tukey-HSD) and, to a greater extent, compared to IDO5 ( P C6/36 v IDO5 = 0.0044, Tukey-HSD) and VeroE6 ( P C6/36 v VeroE6 = 0.0001, Tukey-HSD). Additionally, RVFV produced in CPT-Tert showed higher infectiousness to mosquitoes compared to VeroE6 ( P CPT−tert v VeroE6 = 0.0057, Tukey-HSD), with all other comparisons being not significantly different (Fig. 3 A). The dissemination rate ranges from 12% [6–53] to 75% [35–95] in absence of experiment effect (LR, Chisq = 0.02, Df = 1, P experiment = 0.87) (Fig. 3 B). Dissemination was significantly impacted by dpe (LR, Chisq = 8.7, Df = 1, P dpe = 0.003) but not by RVFV producer cell line (LR, Chisq = 1.7, Df = 4, P stock = 0.77), this trend being conserved across the two experimental replicates (Figure S2F). Overall, RVFV dissemination in Ae. aegypti increased over time, although this was independent of the producer cell line used for RVFV stock production. Transmission rate ranged from 0% [0–37] to 50% [18–81] (Fig. 3 C), with large confidence intervals being driven by the small sample size due to previous infection and dissemination barriers, while no significant experiment effect could be detected (LR, Chisq = 1.1, Df = 1, P experiment = 0.29) (Fig. 3 A-B). The RVFV producer cell line had no impact on the proportion of infected saliva at 21 dpe (LR, Chisq = 7.14, Df = 4, P stock = 0 .12), this result being consistent across the two experiments (Figure S2G). Altogether, these data confirm previous observations showing that RVFV infection rate is time-independent, while dissemination rate increases with dpe. They also show that the producer cell line used impacts Ae. aegypti vector competence for RVFV, with RVFV produced in C6/36 mosquito cells having the highest infectivity for mosquitoes. However, this effect is observed only at the early step of RVFV infection, specifically at the midgut infection stage. Infectious dose in the blood meal is a major driver of Aedes aegypti vector competence for RVFV Previous experiments were conducted with less than 7 log 10 FFU/mL of blood. Rather than relying on a single dose that resulted in 10 to 50% of infected vectors (Figs. 1 and 3 ), it is important to test a range of doses covering the observed viremia levels 19 , 21 . To explore the impact of RVFV dose on vector competence, a dose-response experiment was conducted in Ae. aegypti using RVFV produced in the two cell lines that reached titers exceeding 7 log 10 FFU/mL ( i.e. , MDBK and C6/36). Mosquitoes were exposed to RVFV concentrated at 5.5, 6.5 and 7.5 log 10 FFU/mL (hereafter referred to as low, medium and high dose, respectively) in the blood meal in two experimental replicates. Viral titers remained stable during the blood feeding, regardless of the producer cell line or the experiment (Figure S3A). Due to a small but significant experiment effect (LR, Chisq = 4.38, Df = 1, P experiment = 0.03), the two experiments were analysed separately. Both showed a similar pattern with dose being the only factor significantly impacting the infection rate (LR, Chisq = 4.55, Df = 1, P dose = 0.03 for experiment 1; LR, Chisq = 7.05, Df = 1, P dose = 0.007 for experiment 2) whereas the producer cell line, the dpe or their interaction had no effect (Figure S3B). Accordingly, the two experiments were pooled for analysis, revealing that the infection rate was influenced solely by virus dose (LR, Chisq = 15.9, Df = 1, P dose = 6.55e − 5 ) (Fig. 4 A). Ae. aegypti infection rate increased from 3% [0.5–11] to 84.7% [70–93] as RVFV dose increased in the blood meal (Fig. 4 A). The mean infection rate varied between 3–15%, 28–50% and 55-84.7% for low, medium and high doses, respectively, across RVFV produced in MDBK and C6/36 (Fig. 4 A). Logistic regression analysis showed a sharp increase of RVFV infection between low and high doses, with an oral infectious dose, for 50% of the mosquitoes (OID 50 ), of 7.24 and 6.78 log 10 FFU/mL for RVFV produced in MDBK and C6/36, respectively (Fig. 4 B). The dissemination rate ranged from 0% [0–69] to 94% [80–99] with a high variability due to low sample size especially at the lowest virus dose (Fig. 4 C). Despite no significant experiment effect (LR, Chisq = 0.015, Df = 1, P experiment = 0.89), the full factorial generalized linear model (GLM) did not highlight any significant impact of dose, dpe, producer cell line or their interactions on the dissemination rate. An additive GLM (ignoring potential interactions between dpe, stocks and dose) showed a significant impact of the dpe (LR, Chisq = 58.9, Df = 1, P dpe = 1.58e − 14 ) and, to a lesser extent, of the producer cell line (LR, Chisq = 5.7, Df = 1, P stock = 0.01) and the dose (LR, Chisq = 5.6, Df = 1, P dose = 0.01) on the dissemination rate. When analysing each experiment separately, experiment 1 showed a significant but small effect of the interaction between dpe and producer cell line on the dissemination rate (LR, Chisq = 3.9, Df = 1, P stock*dpe = 0.046), whereas experiment 2 highlighted only an effect of the dpe (LR, Chisq = 27.6, Df = 1, P dose = 1.43e − 7 ) (Figure S3C). Overall, it suggests that dpe is the main driver of RVFV dissemination in Ae. aegypti , with a major increase from 7 to 14 dpe: dissemination rates rose from 15–20% to 72–79% for RVFV produced in MDBK and from 15–53% to 72–73% for RVFV produced in C6/36 at medium and high doses, respectively (Fig. 4 C). However, the above analysis cannot rule out an effect of the virus producer cell line and virus dose on RVFV dissemination, as RVFV produced in C6/36 showed higher dissemination rates than RVFV produced in MDBK at the low dose and early time point (7 dpe), as well as at the high dose and late time point (21 dpe). Transmission rate ranged from 25% [1–78] to 100% [19–100] with large confidence intervals due to previous infection and dissemination barriers, and no detectable experiment effect (LR, Chisq = 2, Df = 1, P experiment = 0.15). As for dissemination, the full factorial GLM did not show any significant impact of the producer cell line nor the virus dose on transmission rate. However, an additive GLM (ignoring the stock x dose interaction) indicated that transmission at 21 dpe varied significantly according to producer cell line (LR, Chisq = 12.2, Df = 1, P stock = 0.0004) (Fig. 4 D). Analysis of each experiment separately showed a small but significant effect of producer cell line on transmission for experiment 2 only (LR, Chisq = 3.9, Df = 1, P stock = 0.048) (Figure S3D). As the sample size was too low to fully capture the impact of producer cell line and dose on RVFV transmission by Ae. aegypti , the transmission efficiency was analysed at 21 dpe. In absence of experiment effect (LR, Chisq = 1.19, Df = 1, P experiment = 0.27), we observed a significant effect of the producer cell line in interaction with the dose on Ae. aegypti TE for RVFV (LR, Chisq = 9.4, Df = 1, P stock x dose = 0.002)(Fig. 4 E). This interaction was significant in experiment 1 (LR, Chisq = 14.6, Df = 1, P stock x dose = 0.0001) but not in experiment 2 (LR, Chisq = 0.24, Df = 1, P stock x dose = 0.6), where only the effect of the dose on TE was significant (LR, Chisq = 23.7, Df = 1, P dose = 1.12e − 6 ) (Figure S3E). These data indicate that Ae. aegypti TE is primarily influenced by RVFV dose, with a smaller contribution from the producer cell line used for RVFV stock preparation. Overall, Ae. aegypti vector competence is mainly driven by RVFV dose in the blood meal during the infection step, regardless of the dpe or producer cell line, and by dpe at the dissemination step. DISCUSSION Understanding species-specific differences in vector competence is critical for predicting RVFV transmission dynamics and identifying potential vector populations in endemic and at-risk regions. While field studies have documented natural RVFV infection in several mosquito species, controlled laboratory experiments with standardized viral doses provide essential mechanistic insights that complement epidemiological observations. Hence, in this work, we used standardized oral mosquito infection assay to explore vector competence (VC) for Phlebovirus riftense (RVFV) reference strain ZH548 in laboratory colonies of Culex and Aedes mosquitoes, the two main vector genera of RVFV worldwide 15 . Our data show that VC for RVFV varies with mosquito genera, virus dose and cell line used to produce virus stock intended for VC assay. Mosquito genotype, virus genotype and virus dose are major determinants of VC for several arboviruses such as dengue virus 39 , 40 . A recent meta-analysis supported the major impact of mosquito species (in collinearity with the genus) and virus titer in the blood meal on VC of Mediterranean Basin mosquito species for RVFV. It also showed stage-specific effects ( e.g. , infection, dissemination, transmission) of additional variables such as mosquito rearing temperature or country of origin on VC 41 . More data is needed to decipher the role of mosquito population (within a given species) and viral lineage on VC for RVFV notably upon standardized oral infection assay with a controlled infectious dose in the blood meal. However, our work highlighted key features of RVFV-mosquito relationship that resonate with other mosquito-arbovirus pathosystems. RVFV ZH548 viral stocks used for mosquito oral exposure were produced on five laboratory cell lines of invertebrate (mosquito) or mammalian (sheep, bovine, monkey) genetic background to mimic vector acquisition of virions from different host origins. We cannot exclude that mutations appeared during virus stock production, in a cell-specific manner, that modified RVFV infectivity for mosquito. But the use of a reverse genetic virus for a limited number of passages (2) combined to the relatively low RVFV mutation rate 42 suggest that other cell-specific host-RVFV interactions might be at play to explain RVFV cell-specific infectivity for mosquitoes. At similar doses, C6/36 (mosquito) and CPT-tert (sheep choroid plexus) derived RVFV showed similar but higher mosquito infection rate compared to stocks from the three other cell lines. At the cellular level, RVFV forms a heterogeneous mix of empty virions and virions with one, two or three segments. Incomplete virions being able to complement each other to allow infection 43 . The C6/36-derived RVFV stocks present a lower viral RNA:infectivity ratio compared to mammalian cells, recapitulating previous results with the RVFV clone 13 vaccine strain 44 . Indeed, the empty virions fraction is lower in mosquito C6/36 (~ 30%) compared to mammalian Vero E6 (~ 50%) cells, with the three RNA segments being more often incorporated within a single virion in C6/36 (23%) compared to Vero E6 (7%) 44 . In addition, the RVFV structural large 78kD protein (LGp), which is incorporated during C6/36 infection but not Vero E6, could promote RVFV infection in the mosquito 45 . Together, it supports a higher infectivity of C6/36 RVFV stocks for mosquitoes compared to Vero E6, although this phenotype does not apply to all mammalian cells, as CPT-tert derived RVFV virions were as infectious for mosquitoes as C6/36 siblings. The enhanced infectivity observed specifically for C6/36 and CPT-tert-derived virions suggests that these cellular environments confer distinct advantages for mosquito infection beyond general cell type differences. While C6/36 benefits from arthropod-specific modifications that may facilitate mosquito cellular recognition, the similar infectivity of CPT-tert virions indicates that specific cellular characteristics rather than simple arthropod versus mammalian distinction drive these effects. This could involve unique lipid compositions, glycosylation patterns, or other post-translational modifications that vary between cell types 46 . Understanding these molecular mechanisms is crucial for developing more accurate cellular models and improve the design of vector-competence laboratory studies. Further studies on RVFV mosquito infectivity could focus on RVFV host target cells such as dendritic cells, macrophages, endothelial, hepatocytes cells 46 , 47 notably at virion’s structural (cryogenic electron microscopy) or biochemical (lipogenomics, glycogenomics) level to reveal the cellular mechanisms governing RVFV host-vector transition. Exposure of viremic patients/animals to mosquitoes could help to decipher the contribution of symptomatic ( e.g. , with neuronal or hepatic infected cells) versus asymptomatic hosts on RVFV acquisition by mosquitoes, with asymptomatic being more infectious to mosquitoes for dengue virus at constant viremia 48 . Interestingly, cell-dependent RVFV stock infectivity was measured only on midgut infection but not on virion escape from midgut cells (dissemination step). Mosquito infection rate by RVFV did not significantly vary over time from day 3 to 21 post-exposure, suggesting that infection must occur quickly prior to blood digestion ( e.g. , within a few hours) as it rapidly triggers viral elimination as suggested for dengue virus 49 . RVFV stocks produced in C6/36 and CPT-tert might be faster and/or more efficient to bind midgut cells, but once the infection established such differences become tenuous. While several potential mammalian RVFV receptors were proposed including heparan sulfate, C-type lectin DC-SIGN or low-density lipoprotein LRP-1, mosquito RVFV receptors remains unknown 50 . Identify cell lines that produce more infectious virions for mosquito is a useful information on the way to mosquito RVFV receptor identification and the design of antiviral strategies. Beyond the effect of RVFV stock cell origin, infectious dose in the blood meal had the strongest impact on mosquito infection rate. Within viral stocks that were available for dose-response experiments ( i.e. , with a sufficient infectious titer) the stock origin did not influence dose-response in mosquito. The oral infectious dose for 50% of the mosquitoes (OID 50 ) ranged from 6.78 to 7.24 log 10 FFU/mL which fits with previous estimates from model-based analysis 51 and ranges within documented host viremia range 52 , 53 . Below 5.63 log 10 FFU/mL of blood, less than 15% of the mosquitoes were infected by RVFV. Based on available animal RVFV viremia profiles, it suggests that only a fraction of hosts is infectious for mosquitoes and for a limited (2–3 days) period 51 . RVFV OID 50 measured in Ae. aegypti can be 100-fold higher compared to Ae. aegypti -dengue virus or Ae. albopictus -chikungunya virus pathosystem 51 , 54 . However, dose-response relationship depends on the complex interaction between mosquito genotype, virus genotype and virus dose which can directly influence arbovirus epidemiology as shown for Zika virus and Ae. aegypti 55 . Therefore, it would be relevant to compare dose-response of field-derived mosquitoes from areas endemic or at risk of RVFV, notably Aedes vexans mosquitoes that are widely-distributed from Africa to the Mediterranean Basin 41 , 56 . Together, mosquito dose-response to RVFV might be an overlooked factor that can explain a significant part of Rift Valley fever epidemiology. Indeed, the high OID 50 values observed for RVFV, combined with the narrow infectious viremia window (2–3 days), suggest that RVFV transmission is more constrained than other major arboviruses, that may explain the episodic nature of outbreaks. Moreover, the observed species-specific vector competence could indicate that regions dominated by different vector species may experience different epidemic patterns and thereby require adapted surveillance strategies. Our results converge towards a major role of midgut infection barrier for RVFV in mosquito, that is mostly circumvented by increasing virus dose in the blood meal. Dissemination of RVFV ZH548 from the midgut to mosquito body cavity was mostly driven by time post-exposure and the mosquito species. Modelling of RVFV intra-vector dynamics 24 provided an added value to our understanding of RVFV-mosquito interaction, showing that RVFV disseminated faster in Cx. quinquefasciatus compare to Ae. aegypti while the latest showed a higher percentage of disseminated individuals. These species-specific differences could arise from distinct anatomical and physiological properties between Aedes and Culex mosquitoes. The faster dissemination in Cx. quinquefasciatus despite lower infection rates suggests different midgut barrier properties, while the higher proportion of disseminated Ae. aegypti indicates more permissive cellular environments once infection is established. These differences may have important implications for transmission dynamics: Cx. quinquefasciatus may contribute to rapid viral amplification during epidemic phases due to faster dissemination, while Ae. aegypti may serve as more efficient reservoir due to its higher overall VC. Mosquito cellular response to viral infection and microbiota have been identified as important drivers of arbovirus dissemination 57 , although studies on RVFV are limited. A study on Culex pipiens exposed to RVFV showed that RNA interference pathway was down-regulated while Toll and Immune deficiency pathways were upregulated early upon an RVFV exposure 58 . RVFV interaction with key mosquito viral factors is currently poorly documented but could influence VC differently depending on the mosquito species considered. This effect can arise from the differential expression between mosquito species of pro- and anti-viral genes, which can modulate viral load and impact dissemination, a minimal viral load threshold being reported to allow dissemination as seen for Zika virus 59 , 60 . This could in turn impact extrinsic incubation period (EIP) that has a major effect on arbovirus basic reproduction number (R 0 ) 61 . A major challenge for VC studies is to leverage the use of experimental infection data ( i.e. , infection, dissemination and transmission at sufficient time points and with enough individuals per time point) for a panel of experimental conditions into DIV models. Here, we modelled RVFV DIV for Aedes or Culex species, but this would be relevant for other VC drivers like virus strain, virus dose or temperature which remain poorly studied for RVFV. The availability of reverse genetic tools for RVFV could help to gain important knowledge on the impact of RVFV mutations on VC, as previously done in mammalian systems (Mehdi Chabert-Ben Cherifa’s thesis). Such screening of mutants in insecta might be worthwhile as RVFV genetic diversity is low overall, suggesting a limited number of candidate mutations with a potential impact on RVFV infection 42 . Climatic variations were also identified as a major driver of RVFV outbreaks worldwide although how temperature impacts mosquito VC for RVFV is not well known 63 . DIV estimation for the above cited VC drivers would allow to test their impact on EIP heterogeneity, in order to integrate it into R 0 modelling that is a major indicator used in risk mitigation strategies. While laboratory-adapted mosquito colonies and standardized conditions may not fully capture wild population diversity and environmental variability, our controlled approach enabled precise identification of key factors modulating RVFV vector competence. The artificial blood feeding method, despite not replicating all host blood component interactions, allowed systematic dose-response analysis that would be difficult to achieve in natural settings. Additionally, although our focus on a single RVFV reverse genetics virus (ZH548) limits direct extrapolation to other viral strains and lineages, it provided essential baseline comparisons across mosquito species and cellular conditions. This study establishes critical foundations for understanding factors that modulate RVFV VC in laboratory settings. The identification of species-specific barriers and DIV, dose-response thresholds and cellular determinants of viral infectivity provides valuable comparative data that complement field studies and offers essential baseline parameters for standardizing future vector competence research and improving experimental reproducibility. Declarations Acknowledgements We thank Anna-Bella Failloux from Institut Pasteur for kindly providing Ae. aegypti colony as well as Grégory L’Ambert and Marie-Laure Setier from EID Méditerranée for providing Cx. quinquefasciatus colony. 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Viruses 17:217 Additional Declarations The authors declare no competing interests. Supplementary Files FigureS1.tiff Vector competence of Aedes aegypti and Culex quinquefasciatus challenged with a RVFV ZH548-RG strain for experiment 1 and 2. Infectious titer of RVFV in blood is calculated at T0 and T1 corresponding of the blood before and after the bloodmeal respectively (A). Dashed lines correspond of accepted variations (A). The proportion of mosquitoes positive for infectious RVFV in the body (B), head (C) and saliva (D) at each day post RVFV exposure (dpe) are represented as proxies of infection, dissemination and transmission rates, respectively. The proportion of mosquitoes engorged (B), positive bodies (C) and positive head (D) are represented above bars. The proportion of individuals with infectious RVFV in the saliva out of the total number of engorged mosquitoes (E) represents a proxy of RVFV transmission efficiency. Vertical bars represent the 95% confidence interval of the proportion (B-E). The size of each dot is proportional to the number of individual mosquitoes tested for RVFV (E). The bars, dots and regression lines are color-coded according to each mosquito species. Panel A aggregates RVFV titer in blood of both experiments. FigureS2.tiff Vector competence of Aedes aegypti challenged with a RVFV ZH548-RG strain produced in five different cell lines for experiment 1 and 2. Infectious titer of RVFV was calculated on two cells type (A). Ratio of genomic titer on infectious titer of RVFV producer cell lines was calculated for stock 1 and 2 then grouped (B). Infectious titer of RVFV in blood is calculated at T0 and T1 corresponding of the blood before and after the bloodmeal, respectively (C). Dashed lines correspond of accepted variations (B). The proportion of mosquitoes positive for infectious RVFV in the body (D), head (F) and saliva (G) at each day post RVFV exposure (dpe) are represented as proxies of infection, dissemination and transmission rates, respectively. As dpe had no impact on infection rate, data from 7, 14 and 21 dpe were grouped to compare RVFV stock infectivity on mosquito (E). The proportion of mosquitoes engorged (D)(E), positive bodies (F) and positive head (G) are represented above bars. Conditions with a similar letter are not significantly different (Tukey-HSD post-hoc test). The number of individual mosquitoes analysed is indicated above each bar. Vertical error bars represent the 95% confidence interval (CI) of the proportion. The cell line used for RVFV stock production is color-coded. FigureS3.tiff Vector competence of Aedes aegypti challenged with three doses of two RVFV ZH548-RG strains produced on MDBK and C6/36 cells in experiment 1 and 2. Infectious titer of RVFV in blood is calculated at T0 and T1 corresponding of the blood before and after the bloodmeal respectively (A). The proportion of mosquitoes positive for infectious RVFV in the body (B), head (C) and saliva (D) at each day post RVFV exposure (dpe) is represented as proxies of infection, dissemination and transmission rates, respectively. The proportion of mosquitoes engorged (B), positive bodies (C) and positive head (D) are represented above bars. The proportion of individuals with infectious RVFV in the saliva out of the total number of engorged mosquitoes (E) is represented as a proxy of RVFV transmission efficiency. The number of individual mosquitoes analysed is indicated above each bar. Vertical error bars or light grey ribbon (for logistic regression lines) represent the 95% confidence interval (CI) of the proportion. The cell line used for RVFV stock production is color-coded. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9196651","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":610454224,"identity":"76f07445-e1f2-4b19-9979-5f562e12b2b3","order_by":0,"name":"Daphné Baudon","email":"","orcid":"https://orcid.org/0009-0007-7416-6627","institution":"IVPC UMR754,EPHE, Université PSL, INRAE, Université Claude Bernard Lyon 1, F-69007, Lyon, France","correspondingAuthor":false,"prefix":"","firstName":"Daphné","middleName":"","lastName":"Baudon","suffix":""},{"id":610454225,"identity":"13dcb05e-af37-46da-8c99-8baf85c42d5c","order_by":1,"name":"Barbara Viginier","email":"","orcid":"https://orcid.org/0000-0002-0733-8974","institution":"IVPC UMR754, EPHE, Université PSL, INRAE, Université Claude Bernard Lyon 1, F-69007, Lyon, France","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Viginier","suffix":""},{"id":610454226,"identity":"43cb57d4-9d75-4221-adc7-da709002f3fa","order_by":2,"name":"Léa Loisel","email":"","orcid":"https://orcid.org/0009-0004-4634-9619","institution":"Oniris, INRAE, BIOEPAR, Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Léa","middleName":"","lastName":"Loisel","suffix":""},{"id":610454227,"identity":"f258c76c-6551-43de-aab1-168f10d75b05","order_by":3,"name":"Marie-Pierre Confort","email":"","orcid":"https://orcid.org/0000-0002-4892-1082","institution":"IVPC UMR754, EPHE, Université PSL, INRAE, Université Claude Bernard Lyon 1, F-69007, Lyon, France","correspondingAuthor":false,"prefix":"","firstName":"Marie-Pierre","middleName":"","lastName":"Confort","suffix":""},{"id":610454228,"identity":"8372352d-0651-4f31-9521-5dc85c5a208f","order_by":4,"name":"Pauline Ezanno","email":"","orcid":"https://orcid.org/0000-0002-0034-8950","institution":"Oniris, INRAE, BIOEPAR, Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"Ezanno","suffix":""},{"id":610454229,"identity":"c6d73ae1-4757-4e3f-b95e-b17cc6fb5910","order_by":5,"name":"Gaël Beaunée","email":"","orcid":"https://orcid.org/0000-0002-0002-2627","institution":"Oniris, INRAE, BIOEPAR, Nantes, France","correspondingAuthor":false,"prefix":"","firstName":"Gaël","middleName":"","lastName":"Beaunée","suffix":""},{"id":610454235,"identity":"632d1b95-8b57-40ff-ae28-0e3ca064f94c","order_by":6,"name":"Maxime Ratinier","email":"","orcid":"https://orcid.org/0000-0003-2271-3549","institution":"IVPC UMR754, EPHE, Université PSL, INRAE, 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Raquin","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6814-4199","institution":"IVPC UMR754, EPHE, Université PSL, INRAE, Université Claude Bernard Lyon 1, F-69007, Lyon, France","correspondingAuthor":true,"prefix":"","firstName":"Vincent","middleName":"","lastName":"Raquin","suffix":""}],"badges":[],"createdAt":"2026-03-23 07:08:07","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9196651/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9196651/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105264821,"identity":"a62683e0-97e8-421e-852f-9c4c59df4d8b","added_by":"auto","created_at":"2026-03-24 07:15:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector competence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAe. aegypti\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCx. quinquefasciatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenged by RVFV ZH548-RG strain.\u003c/strong\u003eThe proportion of mosquitoes positive for infectious RVFV in the body (A), head (B) and saliva (C) at each day post RVFV exposure (dpe) are represented as a proxy of infection, dissemination and transmission rates, respectively. The proportion of mosquitoes engorged (A), positive bodies (B) and positive head (C) are represented above bars. The proportion of individuals with infectious RVFV in the saliva out of the total number of engorged mosquitoes (D) represents a proxy of RVFV transmission efficiency. Vertical bars represent the 95% confidence interval of the proportion (A-C). The size of each dot is proportional to the number of individual mosquitoes tested for RVFV (D). The bars, dots and regression lines are color-coded according to mosquito species, as indicated. Panels A to D aggregate data from two independent experiments (Figure S1).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/ef60ce621bfce07ba4121dd5.png"},{"id":105264822,"identity":"7b233b04-475f-4a10-9cbf-fc0dc47a89c2","added_by":"auto","created_at":"2026-03-24 07:15:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInference of the duration distributions of the intra-vector infection dynamic stages for each species. \u003c/strong\u003eProportion of selected models among those tested: IbetaDexpo, IbetaDbeta, IexpoDexpo, IexpoDbeta (these models differ based on the distribution used – either beta or exponential - for the infected and disseminated stages, as indicated by the first and second parts of their names, respectively) (A). Average of the selected distributions in the infected stage for the main selected model (B). The distributions of stays in states I (B) and D (C) obtained using the most frequently selected model (BetaExpo for \u003cem\u003eAe. aegypti\u003c/em\u003e and ExpoExpo for \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Onlinefloatimage22.png","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/395094ecc3ee18d542937e55.png"},{"id":105564631,"identity":"73eccc54-c886-48be-ba6e-4a8bd54373f3","added_by":"auto","created_at":"2026-03-27 12:50:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector competence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAe. aegypti\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenged with RVFV ZH548-RG strain produced in five different cell lines.\u003c/strong\u003e The proportion of mosquitoes positive for infectious RVFV in the body (A), head (B) and saliva (C) at each day post RVFV exposure (dpe) are represented as proxies of infection, dissemination and transmission rates, respectively. The proportion of mosquitoes engorged (A), positive bodies (B) and positive head (C) are represented above bars. As dpe had no impact on infection rate, data from 7, 14 and 21 dpe were grouped to compare RVFV stock infectivity in mosquito (D). Conditions with a similar letter are not significantly different (Tukey-HSD \u003cem\u003epost-hoc\u003c/em\u003etest). The number of individual mosquitoes analysed is indicated above each bar. Vertical error bars represent the 95% confidence interval (CI) of the proportion. The cell line used for RVFV stock production is color-coded. Panels A to C aggregate data from two independent experiments (Figure S2).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/1082e32e43163ef27176ba0b.png"},{"id":105264824,"identity":"dc380a2e-b78c-420e-8726-b9b0c6a66455","added_by":"auto","created_at":"2026-03-24 07:15:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector competence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAedes aegypti\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenged with the three doses of RVFV ZH548-RG strain produced either in MDBK or C6/36 cells. \u003c/strong\u003eThe proportion of mosquitoes positive for infectious RVFV in the body (A), head (C) and saliva (D) at each day post RVFV exposure (dpe) is represented as proxies of infection, dissemination and transmission rates, respectively. The proportion of individuals with infectious RVFV in the saliva out of the total number of engorged mosquitoes (E) is represented as a proxy of RVFV transmission efficiency. The proportion of mosquitoes engorged (A)(E), positive bodies (C) and positive head (D) are represented above bars. The logistic regression line of mosquito infection rate as a function of RVFV dose in the blood meal is shown (B), with the oral infectious dose required to infect 50% of the mosquitoes (OID\u003csub\u003e50\u003c/sub\u003e) expressed in log\u003csub\u003e10\u003c/sub\u003e (FFU/mL). The number of individual mosquitoes analysed is indicated above each bar. Vertical error bars or light gray ribbon (for logistic regression lines) represent the 95% confidence interval (CI) of the proportion. The cell line used for RVFV stock production is color-coded. Panels A to E aggregate data from two independent experiments (Figure S3).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/61b10b5746f25aaffbce6286.png"},{"id":105569492,"identity":"97287d9a-4e75-4a7d-90fd-48bcc2b58190","added_by":"auto","created_at":"2026-03-27 13:12:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1057746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/5625731c-d9e6-40eb-b9d2-7a3b3ba2ab68.pdf"},{"id":105264825,"identity":"7d5bc1e0-51ed-4540-82ac-f10f98a407ea","added_by":"auto","created_at":"2026-03-24 07:15:13","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16106754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector competence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAedes aegypti\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCulex quinquefasciatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenged with a RVFV ZH548-RG strain for experiment 1 and 2. \u003c/strong\u003eInfectious titer of RVFV in blood is calculated at T0 and T1 corresponding of the blood before and after the bloodmeal respectively (A). Dashed lines correspond of accepted variations (A). The proportion of mosquitoes positive for infectious RVFV in the body (B), head (C) and saliva (D) at each day post RVFV exposure (dpe) are represented as proxies of infection, dissemination and transmission rates, respectively. The proportion of mosquitoes engorged (B), positive bodies (C) and positive head (D) are represented above bars. The proportion of individuals with infectious RVFV in the saliva out of the total number of engorged mosquitoes (E) represents a proxy of RVFV transmission efficiency. Vertical bars represent the 95% confidence interval of the proportion (B-E). The size of each dot is proportional to the number of individual mosquitoes tested for RVFV (E). The bars, dots and regression lines are color-coded according to each mosquito species. Panel A aggregates RVFV titer in blood of both experiments.\u003c/p\u003e","description":"","filename":"FigureS1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/5aa0d38cc271c253debcd9cf.tiff"},{"id":105264826,"identity":"38276c86-8326-4eee-a107-13ffdf7ba3c5","added_by":"auto","created_at":"2026-03-24 07:15:13","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25405430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector competence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAedes aegypti\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenged with a RVFV ZH548-RG strain produced in five different cell lines for experiment 1 and 2. \u003c/strong\u003eInfectious titer of RVFV was calculated on two cells type (A). Ratio of genomic titer on infectious titer of RVFV producer cell lines was calculated for stock 1 and 2 then grouped (B). Infectious titer of RVFV in blood is calculated at T0 and T1 corresponding of the blood before and after the bloodmeal, respectively (C). Dashed lines correspond of accepted variations (B). The proportion of mosquitoes positive for infectious RVFV in the body (D), head (F) and saliva (G) at each day post RVFV exposure (dpe) are represented as proxies of infection, dissemination and transmission rates, respectively. As dpe had no impact on infection rate, data from 7, 14 and 21 dpe were grouped to compare RVFV stock infectivity on mosquito (E). The proportion of mosquitoes engorged (D)(E), positive bodies (F) and positive head (G) are represented above bars. Conditions with a similar letter are not significantly different (Tukey-HSD \u003cem\u003epost-hoc\u003c/em\u003e test). The number of individual mosquitoes analysed is indicated above each bar. Vertical error bars represent the 95% confidence interval (CI) of the proportion. The cell line used for RVFV stock production is color-coded.\u003c/p\u003e","description":"","filename":"FigureS2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/c1104e56f59b263c63256b0d.tiff"},{"id":105564594,"identity":"5de7dbb1-a0a6-4365-8d87-71aa0ab78316","added_by":"auto","created_at":"2026-03-27 12:50:10","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23704826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVector competence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAedes aegypti\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e challenged with three doses of two RVFV ZH548-RG strains produced on MDBK and C6/36 cells in experiment 1 and 2.\u003c/strong\u003e Infectious titer of RVFV in blood is calculated at T0 and T1 corresponding of the blood before and after the bloodmeal respectively (A). The proportion of mosquitoes positive for infectious RVFV in the body (B), head (C) and saliva (D) at each day post RVFV exposure (dpe) is represented as proxies of infection, dissemination and transmission rates, respectively. The proportion of mosquitoes engorged (B), positive bodies (C) and positive head (D) are represented above bars. The proportion of individuals with infectious RVFV in the saliva out of the total number of engorged mosquitoes (E) is represented as a proxy of RVFV transmission efficiency. The number of individual mosquitoes analysed is indicated above each bar. Vertical error bars or light grey ribbon (for logistic regression lines) represent the 95% confidence interval (CI) of the proportion. The cell line used for RVFV stock production is color-coded.\u003c/p\u003e","description":"","filename":"FigureS3.tiff","url":"https://assets-eu.researchsquare.com/files/rs-9196651/v1/c51becb19b69040c24d26f6f.tiff"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eInfluence of biotic factors on Rift Valley Fever virus infection dynamics in mosquitoes\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eArthropod-borne viruses (arboviruses) are transmitted to vertebrate hosts by arthropod vector during blood-feeding. Arboviruses infections account for 28.8% of (re)-emerging viral infectious diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, causing hundreds of millions cases and representing a major public health burden\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Arbovirus transmission is driven by population and individual-level interactions between host, vector and pathogen under the influence of environmental parameters. Establish how variations in these factors impact disease transmission, within areas at risk or endemic for an arbovirus, is a fundamental basis of risk mitigation strategies. For example, the vector species, the virus strain and the viral dose to which the vector is exposed during a blood-meal can modulate vector competence (VC, \u003cem\u003ei.e.\u003c/em\u003e, vector\u0026rsquo;s ability to become infected and then transmit a pathogen), which in turn can influence disease dynamics. However, this has not been thoroughly investigated for several arbovirus-vector pairs such as RVFV in mosquitoes. Moreover, the experimental design (virus production method, vector blood feeding strategy and the number of time points post virus exposure) can hinder our ability to capture the full dynamics of VC notably its time component, the extrinsic incubation period (EIP).\u003c/p\u003e \u003cp\u003eRift Valley Fever virus (RVFV) was first reported in 1930 in Kenya, in the Rift Valley\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and is now widespread in Africa\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e where it has caused hundreds of human deaths, significant livestock losses and major socio-economic impacts over the past 100 years\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Over the past 40 years, RVFV spread to the Indian ocean in 1990\u003csup\u003e10\u003c/sup\u003e then in Arabia Peninsula in 2000\u003csup\u003e11,12\u003c/sup\u003e pushing the World Health Organisation to consider RVFV as a priority pathogen with worldwide spread potential\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. RVFV transmission potential is partly linked to its wide host range, the virus being found a variety of wild animals as well as in livestock, including bovine and ovine. RVFV is also transmitted by large spectrum of vectors mainly mosquitoes of \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e genera, with at least 30 mosquito species being identified as potential vectors\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Some species have been both found positive for RVFV in the field and competent under laboratory conditions including \u003cem\u003eAedes aegypti\u003c/em\u003e, \u003cem\u003eAedes mcintoshi\u003c/em\u003e, \u003cem\u003eAedes vexans\u003c/em\u003e, \u003cem\u003eCulex pipiens\u003c/em\u003e and \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Therefore, they represent relevant targets for RVFV-mosquito studies. As observed for other arboviruses, mosquito VC for RVFV depends on both mosquito and virus genotypes despite this is based on a limited number of studies \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Host viremia level is another key determinant of VC as suggested by direct mosquito feeding on viremic hamsters, although these studies did not allowed to estimate dose-VC relationship\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The duration and amplitude of RVFV viremia in vertebrate hosts varies across species, such as sheep were it ranges from 5 to 7 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL with a peak at day 3 post-infection\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In this context, the use of an artificial blood feeding allows to avoid animals and provides a standardized and reproducible\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e assay. In addition, artificial blood feeding allows to spike the blood meal\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e with a viral stock of controlled genetic composition and cellular origin, via the use of reverse genetics systems and selected amplification cell lines respectively. To our knowledge, the impact of the cell line used for RVFV stock production on VC has not been addressed, and no proper RVFV dose-response experiment was performed upon a range of host viremia-like doses. Recently a stochastic compartmental model of intra-vector infection dynamics (IVD) was developed which, when combined with a tailored experimental design, improves our estimation of VC and EIP distribution\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. While the IVD is critical to characterize the EIP distribution, it has not yet been characterized for RVFV.\u003c/p\u003e \u003cp\u003eThis study aims to estimate VC of a reference RVFV strain (ZH548) produced by reverse genetics from various cell lines for two mosquito laboratory colonies \u003cem\u003e(Ae. aegypti\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e) exposed to host-representative virus doses. Using this approach, a unique dataset was generated demonstrating that the mosquito, the viral dose and the cell line used for viral stocks production influence VC for RVFV although at different steps of the mosquito infection cycle. Estimating RVFV IDV showed that the time required to switch from an infected to a disseminated stage is mosquito-species dependent, providing critical insights for RVFV transmission potential modelling. This work represents an important step towards the understanding of RVFV-mosquito interactions, that will help to improve prediction and mitigation of RVFV (re)emergence worldwide.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eTwo widely used cell lines for RVFV production, namely VeroE6 produced in monkey kidney (kindly provided by ANSES) and C6/36 produced in \u003cem\u003eAe. albopictus\u003c/em\u003e mosquito larvae (European Collection of Authenticated Cell Cultures, reference 89051705), were used. In addition, MDBK, IDO5 and CPT-Tert\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, originating from, bovine kidney, sheep derma and sheep choroid plexus respectively, were also used for RVFV stock production. Mammalian cells were maintained at 37\u0026deg;C, 5% of CO\u003csub\u003e2\u003c/sub\u003e in 75cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e flasks and passaged once a week (1:10 dilution). VeroE6, MDBK and IDO5 were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Sigma) supplemented with 4% (v:v) foetal bovine serum (FBS, Dominique Dutscher), 1% (v:v) penicillin-streptomycin solution (PS, Gicbo) (Penicillin 5U/mL and Streptomycin 5\u0026micro;g/mL) and 1% (v:v) sodium pyruvate. CPT-Tert were cultured in Iscove Modified Dubelcco Madie (Gibco) with 10% FBS and 1% PS. Mosquito C6/36 cells were maintained at 28\u0026deg;C, without CO\u003csub\u003e2\u003c/sub\u003e in 25cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e flasks and passaged weekly (1:10 dilution) in Leibovitz\u0026rsquo;s L-15 Medium (L-15, Gibco) supplemented with 10% of FBS, 10% Tryptose Phosphate Broth (Sigma) and 1% PS.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVirus stock production and titration\u003c/h3\u003e\n\u003cp\u003eThe complete genomic sequence of RVFV ZH548 strain (isolated from a symptomatic child in Egypt in 1977 and belonging to the genetic lineage A\u003csup\u003e26\u003c/sup\u003e) was used to generate a reverse genetic clone (ZH548-RG). Briefly, ZH548 segment L, M and S (consensus sequences were deposited in GenBank with accession numbers OR805805, OR805806, OR805807) were cloned into a pTVT7 plasmid downstream of a T7 promoter. The three plasmids were transfected in hamster kidney cells BSR T7/5 CL21 (kindly provided by Prof. Alain Kohl, Liverpool\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e) to produce an initial virus stock (P0) that was amplified once in VeroE6 cells (P1) and titrated By plaque forming assay using VeroE6 cells (titers are expressed as plaque forming units per mL, PFU/mL)\u003csup\u003e28\u003c/sup\u003e. The P1 stock was then inoculated at a multiplicity of infection of 0.001 on the five cell lines described above to obtain the working virus stock (P2). Briefly, cells were seeded the day prior infection at 5 and 6 log\u003csub\u003e10\u003c/sub\u003e cell per 25-cm\u003csup\u003e2\u003c/sup\u003e flask for mammalian and insect cells, respectively. After 3h, virus suspension was removed and 5 mL of fresh media were added per flask. Infection was stopped upon observation of cytopathic effects (CPE) for mammalian cells (typically between 2- and 4-days post-inoculation) or at 3 days for C6/36 cells (which do not display CPE). Cell supernatants were harvested, centrifugated 4 min at 1000 G to remove cell debris and then stored at -80\u0026deg;C as aliquots. A mock (flask without virus) was also prepared concomitantly as control. Two independent P2 stocks were produced, namely P2-stock1 and P2-stock2. The P2-stock1 and P2-stock2 infectious titers were measured by fluorescent focus assay (FFA) on C6/36 cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The day prior infection, 5.5 log\u003csub\u003e10\u003c/sub\u003e cells/well were seeded into 96-well plates (TPP). After removing cell culture media, 50 \u0026micro;L of a serial dilution (1:10) of each P2 stock were added per well. Plates were incubated for 1 h at 28\u0026deg;C then a 150 \u0026micro;L overlay of a 1:1 (v:v) mix of cell culture media and 3.2% carboxymethyl cellulose solution (Sigma) were added per well. The plates were incubated for 3 days at 28\u0026deg;C, then the cells were fixed using 150 \u0026micro;L/well of a 4% paraformaldehyde solution in 1X Dulbecco\u0026rsquo;s phosphate buffered saline (DPBS, Gibco) for 20 min at room temperature. After 3 washes in 1X DPBS, cells were permeabilized for 30 min at room temperature using 40 \u0026micro;L/well of 0.3% (v:v) Triton X-100 (Sigma) in 1X DPBS\u0026thinsp;+\u0026thinsp;1% Bovine Serum Albumin (BSA, Sigma). After 3 washes in 1X DPBS, 40 \u0026micro;L/well of RVFV anti-N antibody (produced in rabbit), kindly given by Dr Benjamin Brennan (MRC\u0026mdash;University of Glasgow Centre for Virus Research\u0026mdash;Glasgow)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and diluted 1:1000 in 1X DPBS\u0026thinsp;+\u0026thinsp;1% BSA were added in each well as a primary antibody. Cells were incubated for 1h30 at 37\u0026deg;C, rinsed 3 times in 100 \u0026micro;L of 1X DPBS and then incubated for 1 h at 37\u0026deg;C with 40 \u0026micro;L of Alexa546-conjugated anti-rabbit secondary antibody (produced in goat) (ThermoFisher) diluted at 1:2000 in 1X DPBS\u0026thinsp;+\u0026thinsp;1% BSA. Cells were rinsed 3 times in 1X DPBS, once with tap water and stored at 4\u0026deg;C protected from light. Plates were analysed within a week under a Colibri 7 fluorescence microscope (Zeiss) under the 10X objective. The number of fluorescent foci was counted and the infectious titer was expressed as fluorescent focus forming unit per mL (FFU/mL). The absence of viral signal was confirmed in wells inoculated with cell culture media only (negative controls). For the P2-stock1, FFA titration was performed in parallel on C6/36 and VeroE6 cells using the same protocol.\u003c/p\u003e\n\u003ch3\u003eMosquito colonies maintenance\u003c/h3\u003e\n\u003cp\u003eLaboratory colonies of \u003cem\u003eAe. aegypti\u003c/em\u003e PAEA\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (Anna-Bella Failloux, Institut Pasteur Paris) and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e SLAB (Gr\u0026eacute;gory L\u0026rsquo;Ambert and Marie-Laure Setier, EID M\u0026eacute;diterran\u0026eacute;e) were used. Colonies were maintained at IVPC laboratory biosafety level 2 insectary in mass rearing under standard conditions (26\u0026deg;C, 70% relative humidity, 12:12 hours light:dark cycles) using Hemotek membrane feeding system and human blood (from multiple anonymous donors collected by EFS AURA under the CODECOH agreement DC-2019-3507). The \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e colony was maintained continuously (no egg drying) while \u003cem\u003eAe. aegypti\u003c/em\u003e egg papers were stored at 26\u0026deg;C, 70% relative humidity for up to 4 months. Larvae were maintained in 23 x 34 x 7 cm plastic trays (Gilac) with dechlorinated tap water supplemented with a 3:1 (w:w) mix of tropical fish food (Tetramin) and yeast powder (Biover). Adults were maintained in 30 x 30 x 30 cm cages (Bioquip) at 26\u0026deg;C, 70% relative humidity, 12:12 h light:dark cycle with permanent access to 10% (m:v) sugar solution.\u003c/p\u003e\n\u003ch3\u003eMosquito artificial infectious blood meal\u003c/h3\u003e\n\u003cp\u003eThe detailed protocol used here for mosquito artificial blood meal is available regarding \u003cem\u003eAe. aegypti\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, although slight adjustments were done regarding \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e. Fifteen days prior the infectious blood feeding, \u003cem\u003eAe. aegypti\u003c/em\u003e eggs were placed in dechlorinated tap water in a vacuum chamber (-400 mm Hg) at 26\u0026deg;C for ~\u0026thinsp;12h while \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e were offered a blood meal in order to obtain first-instar larvae the day before the infectious blood meal. Synchronized first-instar larvae from both species were then transferred in plastic trays at a density of 200 larvae/tray, with 1.5 L of dechlorinated tap water. Optimal larval development was obtained by adding 0.1 g of food in each tray every two days. The day prior infectious blood meal, 4 to 7-day old females were placed in 136 x 81 mm plastic feeding boxes (Corning-Gosselin) with 45 to 60 females per box and 1 male. Females were transfered in the level 3 biosafety facility (SFR AniRA, Lyon Gerland) at 26\u0026deg;C, 80% of humidity, 12:12h light:dark cycle with no access to sugar solution. A human erythrocyte suspension was prepared the day prior the infectious blood meal. Briefly, human total blood was diluted v:v in PBS, centrifugated 15 min at 1500 G and the supernatant was removed, this operation being performed three times in total. The resulting washed erythrocytes suspension was mixed 2:1 (v:v) with viral suspension, and supplemented with 2% 0.5 M ATP as a phagostimulant (Sigma). Feeders (Hemotek) were covered with pig small intestine and filled with 3 mL of infectious blood mixture. Females were allowed to feed for 20 minutes at 26\u0026deg;C in presence of smelly socks and regular short C0\u003csub\u003e2\u003c/sub\u003e puffs (one every 5 min) to promote feeding. Bloodmeal aliquots were taken before (T0) and immediately after (T\u0026thinsp;+\u0026thinsp;1 hour) the feeding and stored at -80\u0026deg;C for further titration. Mosquitoes were anesthetized on ice (\u003cem\u003eAe. aegypti\u003c/em\u003e) or with C0\u003csub\u003e2\u003c/sub\u003e (\u003cem\u003eCx. quinquefasciatus\u003c/em\u003e) and only fully engorged females were transferred in 1-pint cardboard containers (~\u0026thinsp;15\u0026ndash;25 females/container) with permanent access to cotton soaked with 10% sucrose solution. Cardboard containers were placed in 18 x 18 x 18 inches cages (BioQuip) and incubated at 26\u0026deg;C, 70% humidity, 12:12 h light:dark cycle.\u003c/p\u003e\n\u003ch3\u003eVector competence assays\u003c/h3\u003e\n\u003cp\u003eIndividual mosquitoes and, depending on the experiment, saliva were collected at several days post RVFV exposure (dpe) according to the experimental design. For saliva collection, females were anesthetized as described above then legs and wings were removed. Individuals were placed on a plastic plate maintained by double-sided adhesive tape. The proboscis was inserted in a trimmed 10 \u0026micro;L filtered tip containing 10 \u0026micro;L of FBS and held by modelling clay\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. 2 \u0026micro;L of 1% pilocarpine hydrochloride solution (Sigma) supplemented with 0.1% Tween-20 (Sigma) in water were added on the thorax of each mosquito to promote salivation. Mosquitoes were allowed to salivate at 26\u0026deg;C, 70% relative humidity during 45 minutes. The content of the tip was then expelled in tube filled with 100 \u0026micro;L of DMEM media (Gibco) supplement with 4% FBS and antibiotics (Amphotericin B 2.5\u0026micro;g/mL, Nystatin 10 000 U/mL, Gentamicin, 50\u0026micro;g/mL, Penicillin 5U/mL and Streptomycin 5\u0026micro;g/mL (Gibco)). Of note, saliva samples were deposited immediately (no freezing) on VeroE6 cells in order to maximise the detection of RVFV infectious particles. Following saliva collection, or immediately upon harvest when salivation was not performed, mosquitoes were stored at -80\u0026deg;C. On the day of infectious titration assay, mosquitoes were thawed and individually processed. Mosquito heads and bodies were separated using a pin holder with a 0.15 mm minutien pins (FST). Samples were transferred in individual grinding tubes containing 400 \u0026micro;L of DMEM supplemented with FBS and antibiotics (see above) and one 3-mm diameter tungsten bead (Qiagen). Samples were grinded using a TissueLyzer II (Qiagen) for 2 rounds of 1 min at 30 Hz then centrifugated 5 min at 1000 G and stored at -80\u0026deg;C. RVFV detection was performed by FFA titration assays on VeroE6, as previously described, using 50 \u0026micro;L or 100 \u0026micro;L of raw sample for bodies and heads, respectively, to determine the infectious status (\u003cem\u003ei.e.\u003c/em\u003e, positive or negative for infectious RVFV). Positive (RVFV viral stock) and negative (grinding media) controls were added on each titration plate. This approach allows to estimate vector competence based on three parameters: the infection rate (number of RVFV-positive mosquito bodies / number engorged mosquitoes), the dissemination rate (number of RVFV-positive heads / number of RVFV-positive bodies) and the transmission rate (number of RVFV-positive saliva / number of RVFV-positive heads).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eInfectious titer (expressed as log\u003csub\u003e10\u003c/sub\u003e (FFU/mL)) was considered as continuous response variable and the cell line used for virus production and for virus titration were considered as discrete explanatory variables. Mosquito infection, dissemination and transmission rate were considered as binary response variables (infected or not) while time post virus exposure (dpe) and virus dose in the blood meal were treated as continuous explanatory variables. When several independent experimental replicates were performed, the impact of the experiment effect (discrete variable) on the response variable of interest was tested. If no significant experiment effect was found, data from the different experimental replicates were grouped for downstream analysis. A full factorial linear mixed model (gaussian error and an identity link function) was used to estimate the effect of the cell line used for virus stock production and virus titration on the viral infectious titer while controlling for random variation among the different independent stocks produced. \u003cem\u003ePost-hoc\u003c/em\u003e Tukey-HSD pairwise tests were applied to perform pairwise comparisons between infectious titres between cell lines. A full factorial generalized linear mixed model (GLMM), with a binomial error and logit link function was used to determine the effect of cell line and dpe and their interaction on mosquito infection rate by RVFV, while controlling for random variation among experimental replicates. Despite no significant experiment effect, GLMM could not be ran for other analyses. Therefore, full factorial generalized linear model (GLM) were used to test for the impact of cell line, mosquito species, virus dose and dpe on mosquito infection rate, dissemination rate, transmission rate or transmission efficiency (number of RVFV-positive bodies / number engorged mosquitoes) depending on the experimental design, excluding dpe for transmission rate when transmission was measured only at a single dpe. All the statistical analyses and figures were produced under R environment (RStudio, POSIT) with the main packages \u003cem\u003eggplot2,Tidyverse\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eplyr\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eemmeans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003emultcomp\u003c/em\u003e. Raw data and scripts used in this study are available ().\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModelling RVFV intra-vector dynamics\u003c/h3\u003e\n\u003cp\u003eRVFV intra-vector dynamics was described using a discrete-time stochastic compartmental model (IVD model) with daily time steps, as previously developed and detailed\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This model represents the same processes observed in the vector competence experiments. It includes one compartment for each IVD stage\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Stage \u003cem\u003eE\u003c/em\u003e represents exposed mosquitoes, when the virus is in the digestive tract following a blood meal. Stage \u003cem\u003eI\u003c/em\u003e corresponds to infected mosquitoes, when the virus is present in the midgut cells. Stage \u003cem\u003eD\u003c/em\u003e denotes disseminated mosquitoes, when the virus has spread to the circulatory system. Finally, stage \u003cem\u003eT\u003c/em\u003e refers to transmitter or infectious mosquitoes, marked by the presence of the virus in the saliva. To account for the non-systematic barrier crossing \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, three parameters, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\gamma\\:}_{I},{\\gamma\\:}_{D}\\:and\\:{\\gamma\\:}_{T}\\)\u003c/span\u003e\u003c/span\u003e were introduced to represent the proportions of mosquitoes for which the infection, dissemination, and transmission barriers were crossed, respectively. To model the distributions of durations in the I and D stages, these compartments were subdivided into several sub-compartments representing possible residence times. Mosquitoes were randomly distributed among these sub-compartments according to either an exponential (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\lambda\\:\\)\u003c/span\u003e\u003c/span\u003e) or a beta distribution (parameters \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e), allowing for flexible shapes of time distributions. The model was implemented in R. Each stochastic model replicate, defined by the parameter set (γ_I,γ_D,γ_T,(α_I,β_I) or λ_I,(α_D,β_D) or λ_D ), represented a single vector competence experiment and produced outputs comparable to those obtained in laboratory experiments, namely the numbers of mosquitoes in the infected, disseminated, and transmitter stages at each observed day post exposure (DPE).\u003c/p\u003e\n\u003ch3\u003eModel calibration\u003c/h3\u003e\n\u003cp\u003eModel parameters were inferred using an Approximate Bayesian Computation (ABC) approach with a Sequential Monte Carlo (SMC) sampler \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and model selection \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, following the framework described in \u003csup\u003e24\u003c/sup\u003e and implemented in the R package BRREWABC. This process included the selection of the best model among four options: IbetaDbeta, IbetaDexpo, IexpoDbeta, and IexpoDexpo. These models differ based on the distribution of duration used\u0026mdash;either beta or exponential\u0026mdash;for the infected and disseminated stages, as indicated by the first and second parts of their names, respectively. Depending on the selected model, five or seven parameters were inferred: the three barrier-crossing proportions (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\gamma\\:}_{I},{\\gamma\\:}_{D},{\\gamma\\:}_{T}\\)\u003c/span\u003e\u003c/span\u003e) and the distribution parameters for stages I ((\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\alpha\\:}_{I},{\\beta\\:}_{I}\\)\u003c/span\u003e\u003c/span\u003e) or \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\:\\lambda\\:}_{I}\\)\u003c/span\u003e\u003c/span\u003e) and D ((\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\alpha\\:}_{D},{\\beta\\:}_{D}\\)\u003c/span\u003e\u003c/span\u003e) or \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\lambda\\:}_{D}\\)\u003c/span\u003e\u003c/span\u003e). The ranges of variation of each of inferred parameters and their justifications are presented in supplementary Table SX. Calibration was performed using aggregated data from two experimental replicates with \u003cem\u003eAe. aegypti\u003c/em\u003e (PAEA) and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e (SLAB) females exposed to 6.7 log\u003csub\u003e10\u003c/sub\u003e FFU/mL of RVFV in the blood meal and harvested at 3, 7, 10, 14, and 21 dpe. For each simulated parameter set (particle), distances between observed and simulated values were computed as the sum of squared errors across the four IVD stages. Particles were accepted when all distances were below stage-specific thresholds. Thresholds were adaptively updated at each iteration based on the 90th percentile of accepted distances, and 800 particles were retained per round. This inference yielded parameter sets describing intra-vector viral dynamics in PAEA and SLAB mosquitoes, allowing comparison of residence-time distributions in I and D and their variation between the two species.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRVFV intra-vector dynamics varies according to the mosquito species\u003c/h2\u003e \u003cp\u003eTwo independent experiments were performed in which both \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e females were simultaneously exposed to a blood meal spiked with ~\u0026thinsp;6.7 log\u003csub\u003e10\u003c/sub\u003e FFU/mL of a RVFV ZH548-RG strain. The virus dose was stable in the blood during the time course of the infectious meal (Figure S1A). At the selected days post RVFV exposure (dpe), individual mosquitoes were tested for the presence of infectious virus in the body, head and saliva as a proxy of infection, dissemination and transmission rates, respectively (values shown as % with the associated 95% confidence interval). The infection rate ranged from 21% [12\u0026ndash;34] to 57% [42\u0026ndash;71] with no significant impact of the experiment (LR, Chisq\u0026thinsp;=\u0026thinsp;0.85, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.35). RVFV infection in mosquitoes did not significantly vary according to dpe (LR, Chisq\u0026thinsp;=\u0026thinsp;0.89, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e= 0.34), the mosquito species (LR, Chisq\u0026thinsp;=\u0026thinsp;0.006, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies\u003c/sub\u003e = 0.93) or their interaction (LR, Chisq\u0026thinsp;=\u0026thinsp;0.13, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e = 0.71) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These results were consistent across the two independent experiments (Figure S1B). Among RVFV infected mosquitoes, virus dissemination ranged from 0% [0\u0026ndash;21] to 70% [49\u0026ndash;85] in \u003cem\u003eAe. aegypti\u003c/em\u003e and from 30% [14\u0026ndash;53] to 42% [21\u0026ndash;66] in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) without significant experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;0.94, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.32) (Figure S1C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRVFV dissemination depended on the interaction between dpe and mosquito species (LR, Chisq\u0026thinsp;=\u0026thinsp;6.54, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e= 0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Of note, this interaction was significant for one of the two experimental replicates (LR, Chisq\u0026thinsp;=\u0026thinsp;12.15, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e= 0.0004, experiment 1) but not in the other (LR, Chisq\u0026thinsp;=\u0026thinsp;0.07, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e = 0.79, experiment 2), where only the dpe significantly impacted dissemination (LR, Chisq\u0026thinsp;=\u0026thinsp;5.05, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e = 0.02, experiment 2) (Figure S1C). Overall, RVFV dissemination increases over time, although this increase depends on the mosquito species, indicating a vector-specific dynamic of RVFV dissemination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe proportion of saliva with infectious RVFV virus ranged from 11% [0.005\u0026ndash;0.49] to 83% [36\u0026ndash;99] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), the large confidence intervals being driven by the small sample size, due to previous infection and dissemination barriers, in absence of experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;0.04, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.82) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). The impact of the interaction between species and dpe on transmission rate was bearly significant (LR, Chisq\u0026thinsp;=\u0026thinsp;3.87, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e = 0.049) when analysing the two experiment together. However, this interaction was not significant for either of the experiments separately (LR, Chisq\u0026thinsp;=\u0026thinsp;0.007, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e = 0.93 for experiment 1 ; Chisq\u0026thinsp;=\u0026thinsp;3.57, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e = 0.058 for experiment 2), while the effect of dpe remained significant in both experiments (LR, Chisq\u0026thinsp;=\u0026thinsp;5.66, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e = 0.01 for experiment 1 ; Chisq\u0026thinsp;=\u0026thinsp;3.93, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e = 0.04 for experiment 2) in absence of species effect (LR, Chisq\u0026thinsp;=\u0026thinsp;0.76, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies\u003c/sub\u003e = 0.39 for experiment 1 ; Chisq\u0026thinsp;=\u0026thinsp;2.46, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies\u003c/sub\u003e = 0.11 for experiment 2) (Figure S1D). Altogether, these data show that RVFV transmission is likely driven by the dpe, with the proportion of infectious mosquitoes increasing over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe transmission efficiency (TE, proportion of mosquitoes with RVFV in the saliva out of the total number of engorged specimens), which encompasses previous infection, dissemination and transmission steps to provide a proxy of RVFV transmissibility by the vector, was calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Results indicated that TE depends on the interaction between dpe and mosquito species (LR, Chisq\u0026thinsp;=\u0026thinsp;11.1, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003especies*dpe\u003c/sub\u003e = 0.0008), a result that is consistent across experiments (Figure S1E). This result indicates that, beyond experimental variations, \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e are stably infected by RVFV in the midgut, allowing for increasing viral dissemination over time, which peaks after 14 dpe. However, vector competence vary according to the species, with \u003cem\u003eAe. aegypti\u003c/em\u003e being overall more competent than \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e for RVFV ZH548-RG strain, likely due to a higher dissemination rate.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo further explore RVFV intra-vector dynamics (IVD) in mosquitoes, including the EIP, our recently developed modelling tool\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e was used to estimate RVFV IVD in \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e using the vector competence data presented above (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Model selection step showed that the estimation of infection (I) and dissemination (D) step durations best fit a beta (I) / exponential (D) distribution for \u003cem\u003eAe. aegypti\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, for \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e, both exponential (I) / exponential (D) and, to a lesser extent, exponential (I) / beta (D) fitted the observed distribution. Using the best fit for both species, the model suggested that the majority of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes spent more time in (I) with a peak at ~\u0026thinsp;10 days compared to \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e for which most of the individuals left (I) in fewer than 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Once in (D), the time spent at this stage was comparable for both species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Overall, vector competence and IVD estimation for the two mosquito species showed that \u003cem\u003eAe. aegypti\u003c/em\u003e has a higher dissemination rate but takes longer to disseminate RVFV compared to \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell line used for RVFV stock production impacts the early stages of mosquito infection\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eAe. aegypti\u003c/em\u003e mosquito population was chosen for subsequent experiments, as it displayed a higher vector competence overall for our RVFV ZH548-RG strain compared to \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We now aim to investigate the impact of the virus-producing cell line on vector competence. Stocks of ZH548-RG strain were produced using five different cell lines (VeroE6 and C6/36, MDBK, CPT-Tert and IDO5) reaching 7\u0026ndash;8 log\u003csub\u003e10\u003c/sub\u003e FFU/mL, regardless of whether VeroE6 or C6/36 was used for virus titration (Figure S2A). No significant difference in viral titer was observed between the two cell lines used for it. Viral RNA:infectivity ratio was calculated and shown that C6/36-derived RVFV stocks present a lower ratio compared to mammalian cells (Figure S2B). Virus doses in the blood meal did not vary according to time or producer cell line, remaining at ~\u0026thinsp;6.5 log\u003csub\u003e10\u003c/sub\u003e FFU/mL in both experiments (Figure S2C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe infection rate ranged from 5% [1\u0026ndash;13] to 34% [22\u0026ndash;48] with no impact of the dpe while controlling for experiment effect (GLMM) (Wald χ\u003csup\u003e2\u003c/sup\u003e, Chisq\u0026thinsp;=\u0026thinsp;1.10, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e = 0.29) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD-E). However, the producer cell line used significantly influenced mosquito infection rate (Wald χ\u003csup\u003e2\u003c/sup\u003e, Chisq\u0026thinsp;=\u0026thinsp;18.5, Df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock\u003c/sub\u003e = 0.0009). As mosquito infection rate did not depend on the dpe, data from 7, 14 and 21 dpe were grouped to interrogate the impact of the RVFV producer cell line on the infection rate while controlling for experiment effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). \u003cem\u003ePost-hoc\u003c/em\u003e tests showed that RVFV produced in C6/36 had a higher infection rate compared to MDBK (\u003cem\u003eP\u003c/em\u003e\u003csub\u003eC6/36 \u003cem\u003ev\u003c/em\u003e MDBK\u003c/sub\u003e = 0.046, Tukey-HSD) and, to a greater extent, compared to IDO5 (\u003cem\u003eP\u003c/em\u003e\u003csub\u003eC6/36 \u003cem\u003ev\u003c/em\u003e IDO5\u003c/sub\u003e = 0.0044, Tukey-HSD) and VeroE6 (\u003cem\u003eP\u003c/em\u003e\u003csub\u003eC6/36 \u003cem\u003ev\u003c/em\u003e VeroE6\u003c/sub\u003e = 0.0001, Tukey-HSD). Additionally, RVFV produced in CPT-Tert showed higher infectiousness to mosquitoes compared to VeroE6 (\u003cem\u003eP\u003c/em\u003e\u003csub\u003eCPT\u0026minus;tert \u003cem\u003ev\u003c/em\u003e VeroE6\u003c/sub\u003e = 0.0057, Tukey-HSD), with all other comparisons being not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe dissemination rate ranges from 12% [6\u0026ndash;53] to 75% [35\u0026ndash;95] in absence of experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;0.02, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.87) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Dissemination was significantly impacted by dpe (LR, Chisq\u0026thinsp;=\u0026thinsp;8.7, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e = 0.003) but not by RVFV producer cell line (LR, Chisq\u0026thinsp;=\u0026thinsp;1.7, Df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock\u003c/sub\u003e = 0.77), this trend being conserved across the two experimental replicates (Figure S2F). Overall, RVFV dissemination in \u003cem\u003eAe. aegypti\u003c/em\u003e increased over time, although this was independent of the producer cell line used for RVFV stock production.\u003c/p\u003e \u003cp\u003eTransmission rate ranged from 0% [0\u0026ndash;37] to 50% [18\u0026ndash;81] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with large confidence intervals being driven by the small sample size due to previous infection and dissemination barriers, while no significant experiment effect could be detected (LR, Chisq\u0026thinsp;=\u0026thinsp;1.1, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.29) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). The RVFV producer cell line had no impact on the proportion of infected saliva at 21 dpe (LR, Chisq\u0026thinsp;=\u0026thinsp;7.14, Df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock\u003c/sub\u003e = 0 .12), this result being consistent across the two experiments (Figure S2G). Altogether, these data confirm previous observations showing that RVFV infection rate is time-independent, while dissemination rate increases with dpe. They also show that the producer cell line used impacts \u003cem\u003eAe. aegypti\u003c/em\u003e vector competence for RVFV, with RVFV produced in C6/36 mosquito cells having the highest infectivity for mosquitoes. However, this effect is observed only at the early step of RVFV infection, specifically at the midgut infection stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInfectious dose in the blood meal is a major driver of\u003c/b\u003e \u003cb\u003eAedes aegypti\u003c/b\u003e \u003cb\u003evector competence for RVFV\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious experiments were conducted with less than 7 log\u003csub\u003e10\u003c/sub\u003e FFU/mL of blood. Rather than relying on a single dose that resulted in 10 to 50% of infected vectors (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e), it is important to test a range of doses covering the observed viremia levels\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To explore the impact of RVFV dose on vector competence, a dose-response experiment was conducted in \u003cem\u003eAe. aegypti\u003c/em\u003e using RVFV produced in the two cell lines that reached titers exceeding 7 log\u003csub\u003e10\u003c/sub\u003e FFU/mL (\u003cem\u003ei.e.\u003c/em\u003e, MDBK and C6/36). Mosquitoes were exposed to RVFV concentrated at 5.5, 6.5 and 7.5 log\u003csub\u003e10\u003c/sub\u003e FFU/mL (hereafter referred to as low, medium and high dose, respectively) in the blood meal in two experimental replicates. Viral titers remained stable during the blood feeding, regardless of the producer cell line or the experiment (Figure S3A). Due to a small but significant experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;4.38, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.03), the two experiments were analysed separately. Both showed a similar pattern with dose being the only factor significantly impacting the infection rate (LR, Chisq\u0026thinsp;=\u0026thinsp;4.55, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edose\u003c/sub\u003e = 0.03 for experiment 1; LR, Chisq\u0026thinsp;=\u0026thinsp;7.05, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edose\u003c/sub\u003e = 0.007 for experiment 2) whereas the producer cell line, the dpe or their interaction had no effect (Figure S3B). Accordingly, the two experiments were pooled for analysis, revealing that the infection rate was influenced solely by virus dose (LR, Chisq\u0026thinsp;=\u0026thinsp;15.9, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edose\u003c/sub\u003e = 6.55e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). \u003cem\u003eAe. aegypti\u003c/em\u003e infection rate increased from 3% [0.5\u0026ndash;11] to 84.7% [70\u0026ndash;93] as RVFV dose increased in the blood meal (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The mean infection rate varied between 3\u0026ndash;15%, 28\u0026ndash;50% and 55-84.7% for low, medium and high doses, respectively, across RVFV produced in MDBK and C6/36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Logistic regression analysis showed a sharp increase of RVFV infection between low and high doses, with an oral infectious dose, for 50% of the mosquitoes (OID\u003csub\u003e50\u003c/sub\u003e), of 7.24 and 6.78 log\u003csub\u003e10\u003c/sub\u003e FFU/mL for RVFV produced in MDBK and C6/36, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe dissemination rate ranged from 0% [0\u0026ndash;69] to 94% [80\u0026ndash;99] with a high variability due to low sample size especially at the lowest virus dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Despite no significant experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;0.015, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.89), the full factorial generalized linear model (GLM) did not highlight any significant impact of dose, dpe, producer cell line or their interactions on the dissemination rate. An additive GLM (ignoring potential interactions between dpe, stocks and dose) showed a significant impact of the dpe (LR, Chisq\u0026thinsp;=\u0026thinsp;58.9, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edpe\u003c/sub\u003e = 1.58e\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e) and, to a lesser extent, of the producer cell line (LR, Chisq\u0026thinsp;=\u0026thinsp;5.7, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock\u003c/sub\u003e = 0.01) and the dose (LR, Chisq\u0026thinsp;=\u0026thinsp;5.6, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edose\u003c/sub\u003e = 0.01) on the dissemination rate. When analysing each experiment separately, experiment 1 showed a significant but small effect of the interaction between dpe and producer cell line on the dissemination rate (LR, Chisq\u0026thinsp;=\u0026thinsp;3.9, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock*dpe\u003c/sub\u003e = 0.046), whereas experiment 2 highlighted only an effect of the dpe (LR, Chisq\u0026thinsp;=\u0026thinsp;27.6, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edose\u003c/sub\u003e = 1.43e\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e) (Figure S3C). Overall, it suggests that dpe is the main driver of RVFV dissemination in \u003cem\u003eAe. aegypti\u003c/em\u003e, with a major increase from 7 to 14 dpe: dissemination rates rose from 15\u0026ndash;20% to 72\u0026ndash;79% for RVFV produced in MDBK and from 15\u0026ndash;53% to 72\u0026ndash;73% for RVFV produced in C6/36 at medium and high doses, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, the above analysis cannot rule out an effect of the virus producer cell line and virus dose on RVFV dissemination, as RVFV produced in C6/36 showed higher dissemination rates than RVFV produced in MDBK at the low dose and early time point (7 dpe), as well as at the high dose and late time point (21 dpe).\u003c/p\u003e \u003cp\u003eTransmission rate ranged from 25% [1\u0026ndash;78] to 100% [19\u0026ndash;100] with large confidence intervals due to previous infection and dissemination barriers, and no detectable experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;2, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.15). As for dissemination, the full factorial GLM did not show any significant impact of the producer cell line nor the virus dose on transmission rate. However, an additive GLM (ignoring the stock \u003cem\u003ex\u003c/em\u003e dose interaction) indicated that transmission at 21 dpe varied significantly according to producer cell line (LR, Chisq\u0026thinsp;=\u0026thinsp;12.2, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock\u003c/sub\u003e = 0.0004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Analysis of each experiment separately showed a small but significant effect of producer cell line on transmission for experiment 2 only (LR, Chisq\u0026thinsp;=\u0026thinsp;3.9, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock\u003c/sub\u003e = 0.048) (Figure S3D). As the sample size was too low to fully capture the impact of producer cell line and dose on RVFV transmission by \u003cem\u003eAe. aegypti\u003c/em\u003e, the transmission efficiency was analysed at 21 dpe. In absence of experiment effect (LR, Chisq\u0026thinsp;=\u0026thinsp;1.19, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eexperiment\u003c/sub\u003e = 0.27), we observed a significant effect of the producer cell line in interaction with the dose on \u003cem\u003eAe. aegypti\u003c/em\u003e TE for RVFV (LR, Chisq\u0026thinsp;=\u0026thinsp;9.4, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock \u003cem\u003ex\u003c/em\u003e dose\u003c/sub\u003e = 0.002)(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). This interaction was significant in experiment 1 (LR, Chisq\u0026thinsp;=\u0026thinsp;14.6, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock \u003cem\u003ex\u003c/em\u003e dose\u003c/sub\u003e = 0.0001) but not in experiment 2 (LR, Chisq\u0026thinsp;=\u0026thinsp;0.24, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003estock \u003cem\u003ex\u003c/em\u003e dose\u003c/sub\u003e = 0.6), where only the effect of the dose on TE was significant (LR, Chisq\u0026thinsp;=\u0026thinsp;23.7, Df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u003csub\u003edose\u003c/sub\u003e = 1.12e\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) (Figure S3E). These data indicate that \u003cem\u003eAe. aegypti\u003c/em\u003e TE is primarily influenced by RVFV dose, with a smaller contribution from the producer cell line used for RVFV stock preparation. Overall, \u003cem\u003eAe. aegypti\u003c/em\u003e vector competence is mainly driven by RVFV dose in the blood meal during the infection step, regardless of the dpe or producer cell line, and by dpe at the dissemination step.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eUnderstanding species-specific differences in vector competence is critical for predicting RVFV transmission dynamics and identifying potential vector populations in endemic and at-risk regions. While field studies have documented natural RVFV infection in several mosquito species, controlled laboratory experiments with standardized viral doses provide essential mechanistic insights that complement epidemiological observations. Hence, in this work, we used standardized oral mosquito infection assay to explore vector competence (VC) for \u003cem\u003ePhlebovirus riftense\u003c/em\u003e (RVFV) reference strain ZH548 in laboratory colonies of \u003cem\u003eCulex\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e mosquitoes, the two main vector genera of RVFV worldwide\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our data show that VC for RVFV varies with mosquito genera, virus dose and cell line used to produce virus stock intended for VC assay. Mosquito genotype, virus genotype and virus dose are major determinants of VC for several arboviruses such as dengue virus\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. A recent meta-analysis supported the major impact of mosquito species (in collinearity with the genus) and virus titer in the blood meal on VC of Mediterranean Basin mosquito species for RVFV. It also showed stage-specific effects (\u003cem\u003ee.g.\u003c/em\u003e, infection, dissemination, transmission) of additional variables such as mosquito rearing temperature or country of origin on VC\u003csup\u003e41\u003c/sup\u003e. More data is needed to decipher the role of mosquito population (within a given species) and viral lineage on VC for RVFV notably upon standardized oral infection assay with a controlled infectious dose in the blood meal. However, our work highlighted key features of RVFV-mosquito relationship that resonate with other mosquito-arbovirus pathosystems.\u003c/p\u003e \u003cp\u003eRVFV ZH548 viral stocks used for mosquito oral exposure were produced on five laboratory cell lines of invertebrate (mosquito) or mammalian (sheep, bovine, monkey) genetic background to mimic vector acquisition of virions from different host origins. We cannot exclude that mutations appeared during virus stock production, in a cell-specific manner, that modified RVFV infectivity for mosquito. But the use of a reverse genetic virus for a limited number of passages (2) combined to the relatively low RVFV mutation rate\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e suggest that other cell-specific host-RVFV interactions might be at play to explain RVFV cell-specific infectivity for mosquitoes. At similar doses, C6/36 (mosquito) and CPT-tert (sheep choroid plexus) derived RVFV showed similar but higher mosquito infection rate compared to stocks from the three other cell lines. At the cellular level, RVFV forms a heterogeneous mix of empty virions and virions with one, two or three segments. Incomplete virions being able to complement each other to allow infection\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The C6/36-derived RVFV stocks present a lower viral RNA:infectivity ratio compared to mammalian cells, recapitulating previous results with the RVFV clone 13 vaccine strain\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Indeed, the empty virions fraction is lower in mosquito C6/36 (~\u0026thinsp;30%) compared to mammalian Vero E6 (~\u0026thinsp;50%) cells, with the three RNA segments being more often incorporated within a single virion in C6/36 (23%) compared to Vero E6 (7%)\u003csup\u003e44\u003c/sup\u003e. In addition, the RVFV structural large 78kD protein (LGp), which is incorporated during C6/36 infection but not Vero E6, could promote RVFV infection in the mosquito\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Together, it supports a higher infectivity of C6/36 RVFV stocks for mosquitoes compared to Vero E6, although this phenotype does not apply to all mammalian cells, as CPT-tert derived RVFV virions were as infectious for mosquitoes as C6/36 siblings. The enhanced infectivity observed specifically for C6/36 and CPT-tert-derived virions suggests that these cellular environments confer distinct advantages for mosquito infection beyond general cell type differences. While C6/36 benefits from arthropod-specific modifications that may facilitate mosquito cellular recognition, the similar infectivity of CPT-tert virions indicates that specific cellular characteristics rather than simple arthropod versus mammalian distinction drive these effects. This could involve unique lipid compositions, glycosylation patterns, or other post-translational modifications that vary between cell types\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Understanding these molecular mechanisms is crucial for developing more accurate cellular models and improve the design of vector-competence laboratory studies. Further studies on RVFV mosquito infectivity could focus on RVFV host target cells such as dendritic cells, macrophages, endothelial, hepatocytes cells\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e notably at virion\u0026rsquo;s structural (cryogenic electron microscopy) or biochemical (lipogenomics, glycogenomics) level to reveal the cellular mechanisms governing RVFV host-vector transition. Exposure of viremic patients/animals to mosquitoes could help to decipher the contribution of symptomatic (\u003cem\u003ee.g.\u003c/em\u003e, with neuronal or hepatic infected cells) \u003cem\u003eversus\u003c/em\u003e asymptomatic hosts on RVFV acquisition by mosquitoes, with asymptomatic being more infectious to mosquitoes for dengue virus at constant viremia\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Interestingly, cell-dependent RVFV stock infectivity was measured only on midgut infection but not on virion escape from midgut cells (dissemination step). Mosquito infection rate by RVFV did not significantly vary over time from day 3 to 21 post-exposure, suggesting that infection must occur quickly prior to blood digestion (\u003cem\u003ee.g.\u003c/em\u003e, within a few hours) as it rapidly triggers viral elimination as suggested for dengue virus\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. RVFV stocks produced in C6/36 and CPT-tert might be faster and/or more efficient to bind midgut cells, but once the infection established such differences become tenuous. While several potential mammalian RVFV receptors were proposed including heparan sulfate, C-type lectin DC-SIGN or low-density lipoprotein LRP-1, mosquito RVFV receptors remains unknown\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Identify cell lines that produce more infectious virions for mosquito is a useful information on the way to mosquito RVFV receptor identification and the design of antiviral strategies.\u003c/p\u003e \u003cp\u003eBeyond the effect of RVFV stock cell origin, infectious dose in the blood meal had the strongest impact on mosquito infection rate. Within viral stocks that were available for dose-response experiments (\u003cem\u003ei.e.\u003c/em\u003e, with a sufficient infectious titer) the stock origin did not influence dose-response in mosquito. The oral infectious dose for 50% of the mosquitoes (OID\u003csub\u003e50\u003c/sub\u003e) ranged from 6.78 to 7.24 log\u003csub\u003e10\u003c/sub\u003e FFU/mL which fits with previous estimates from model-based analysis\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and ranges within documented host viremia range\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Below 5.63 log\u003csub\u003e10\u003c/sub\u003e FFU/mL of blood, less than 15% of the mosquitoes were infected by RVFV. Based on available animal RVFV viremia profiles, it suggests that only a fraction of hosts is infectious for mosquitoes and for a limited (2\u0026ndash;3 days) period\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. RVFV OID\u003csub\u003e50\u003c/sub\u003e measured in \u003cem\u003eAe. aegypti\u003c/em\u003e can be 100-fold higher compared to \u003cem\u003eAe. aegypti\u003c/em\u003e-dengue virus or \u003cem\u003eAe. albopictus\u003c/em\u003e-chikungunya virus pathosystem\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, dose-response relationship depends on the complex interaction between mosquito genotype, virus genotype and virus dose which can directly influence arbovirus epidemiology as shown for Zika virus and \u003cem\u003eAe. aegypti\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Therefore, it would be relevant to compare dose-response of field-derived mosquitoes from areas endemic or at risk of RVFV, notably \u003cem\u003eAedes vexans\u003c/em\u003e mosquitoes that are widely-distributed from Africa to the Mediterranean Basin\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Together, mosquito dose-response to RVFV might be an overlooked factor that can explain a significant part of Rift Valley fever epidemiology. Indeed, the high OID\u003csub\u003e50\u003c/sub\u003e values observed for RVFV, combined with the narrow infectious viremia window (2\u0026ndash;3 days), suggest that RVFV transmission is more constrained than other major arboviruses, that may explain the episodic nature of outbreaks. Moreover, the observed species-specific vector competence could indicate that regions dominated by different vector species may experience different epidemic patterns and thereby require adapted surveillance strategies.\u003c/p\u003e \u003cp\u003eOur results converge towards a major role of midgut infection barrier for RVFV in mosquito, that is mostly circumvented by increasing virus dose in the blood meal. Dissemination of RVFV ZH548 from the midgut to mosquito body cavity was mostly driven by time post-exposure and the mosquito species. Modelling of RVFV intra-vector dynamics\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e provided an added value to our understanding of RVFV-mosquito interaction, showing that RVFV disseminated faster in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e compare to \u003cem\u003eAe. aegypti\u003c/em\u003e while the latest showed a higher percentage of disseminated individuals. These species-specific differences could arise from distinct anatomical and physiological properties between \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e mosquitoes. The faster dissemination in \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e despite lower infection rates suggests different midgut barrier properties, while the higher proportion of disseminated \u003cem\u003eAe. aegypti\u003c/em\u003e indicates more permissive cellular environments once infection is established. These differences may have important implications for transmission dynamics: \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e may contribute to rapid viral amplification during epidemic phases due to faster dissemination, while \u003cem\u003eAe. aegypti\u003c/em\u003e may serve as more efficient reservoir due to its higher overall VC. Mosquito cellular response to viral infection and microbiota have been identified as important drivers of arbovirus dissemination\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, although studies on RVFV are limited. A study on \u003cem\u003eCulex pipiens\u003c/em\u003e exposed to RVFV showed that RNA interference pathway was down-regulated while Toll and Immune deficiency pathways were upregulated early upon an RVFV exposure\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. RVFV interaction with key mosquito viral factors is currently poorly documented but could influence VC differently depending on the mosquito species considered. This effect can arise from the differential expression between mosquito species of pro- and anti-viral genes, which can modulate viral load and impact dissemination, a minimal viral load threshold being reported to allow dissemination as seen for Zika virus\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. This could in turn impact extrinsic incubation period (EIP) that has a major effect on arbovirus basic reproduction number (R\u003csub\u003e0\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. A major challenge for VC studies is to leverage the use of experimental infection data (\u003cem\u003ei.e.\u003c/em\u003e, infection, dissemination and transmission at sufficient time points and with enough individuals per time point) for a panel of experimental conditions into DIV models. Here, we modelled RVFV DIV for \u003cem\u003eAedes\u003c/em\u003e or \u003cem\u003eCulex\u003c/em\u003e species, but this would be relevant for other VC drivers like virus strain, virus dose or temperature which remain poorly studied for RVFV. The availability of reverse genetic tools for RVFV could help to gain important knowledge on the impact of RVFV mutations on VC, as previously done in mammalian systems (Mehdi Chabert-Ben Cherifa\u0026rsquo;s thesis). Such screening of mutants \u003cem\u003ein insecta\u003c/em\u003e might be worthwhile as RVFV genetic diversity is low overall, suggesting a limited number of candidate mutations with a potential impact on RVFV infection\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Climatic variations were also identified as a major driver of RVFV outbreaks worldwide although how temperature impacts mosquito VC for RVFV is not well known\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. DIV estimation for the above cited VC drivers would allow to test their impact on EIP heterogeneity, in order to integrate it into R\u003csub\u003e0\u003c/sub\u003e modelling that is a major indicator used in risk mitigation strategies. While laboratory-adapted mosquito colonies and standardized conditions may not fully capture wild population diversity and environmental variability, our controlled approach enabled precise identification of key factors modulating RVFV vector competence. The artificial blood feeding method, despite not replicating all host blood component interactions, allowed systematic dose-response analysis that would be difficult to achieve in natural settings. Additionally, although our focus on a single RVFV reverse genetics virus (ZH548) limits direct extrapolation to other viral strains and lineages, it provided essential baseline comparisons across mosquito species and cellular conditions. This study establishes critical foundations for understanding factors that modulate RVFV VC in laboratory settings. The identification of species-specific barriers and DIV, dose-response thresholds and cellular determinants of viral infectivity provides valuable comparative data that complement field studies and offers essential baseline parameters for standardizing future vector competence research and improving experimental reproducibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Anna-Bella Failloux from Institut Pasteur for kindly providing \u003cem\u003eAe. aegypti\u003c/em\u003e colony as well as Gr\u0026eacute;gory L\u0026rsquo;Ambert and Marie-Laure Setier from EID M\u0026eacute;diterran\u0026eacute;e for providing \u003cem\u003eCx. quinquefasciatus\u003c/em\u003e colony. We thank Alain Kohl and Ben Brennan from the Liverpool School of Tropical Medecine and the MRC-University of Glasgow Centre for Virus Research respectively for plasmids and cell lines. We acknowledge the contributions of the CELPHEDIA Infrastructure (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.celphedia.eu/\u003c/span\u003e\u003cspan address=\"http://www.celphedia.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), especially the center AniRA in Lyon. This work was supported by the INRAE metaprogram Digit-Bio (MIDIIVEC project), ED 472 EPHE for DB PhD program and the EquipEx+ InfectioTron within the program Investissements d\u0026rsquo;Avenir (ANR-21-ESRE-0023) operated by the French National Research Agency.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJones KE et al (2008) Global trends in emerging infectious diseases. Nature 451:990\u0026ndash;993\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadewell ZJ (2020) Arboviruses and Their Vectors. 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Preprint at. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2024.04.03.587352\u003c/span\u003e\u003cspan address=\"10.1101/2024.04.03.587352\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzerigyik FA, Cagle SM, Wilson WC, Mitzel DN, Kading RC (2025) The Temperature-Associated Effects of Rift Valley Fever Virus Infections in Mosquitoes and Climate-Driven Epidemics: A Review. Viruses 17:217\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"081c8e5b-4ee4-4196-a207-9e7d0842001c","identifier":"10.13039/501100006488","name":"Institut National de la Recherche Agronomique","awardNumber":"MIDIIVEC2022","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"École Pratique des Hautes Études","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rift Valley fever virus, arbovirus, mosquito, vector competence, DIV, Aedes aegypti, Culex quinquefasciatus","lastPublishedDoi":"10.21203/rs.3.rs-9196651/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9196651/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRift 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 its large vector species spectrum. Understanding how genetic and environmental (a)biotic factors shape RVFV transmission by mosquitoes is therefore critical to prevent Rift Valley fever emergence and spread. Studies often focused on main vector competence (VC) drivers such as mosquitoes species or virus dose, for arboviruses currently considered as major human threats worldwide like dengue, chikungunya or Zika viruses. Other potential VC drivers have been overlooked, like the cellular origin of viruses used in VC assays, while some mosquito-borne viruses remain understudied including RVFV. In addition, intra-vector infection dynamics (IVD), represented by the extrinsic incubation period (EIP) distribution within the mosquito population, remains a black box for many vector-arbovirus pairs. Here, we solved some of these gaps by feeding \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e mosquitoes with the reference RVFV ZH548 strain prior to measure viral infection, dissemination and transmission in individual mosquitoes and estimate RVFV IVD. Major VC variations were observed according to mosquito, virus dose and cell line used for virus stock production together with key differences in IVD between \u003cem\u003eAe. aegypti\u003c/em\u003e and \u003cem\u003eCx\u003c/em\u003e. \u003cem\u003equinquefasciatus\u003c/em\u003e. This study provides a reference data set of mosquito VC for RVFV, for a range of host-like virus doses and stocks, including the EIP range for the two major RVFV vector genera (\u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e). Altogether, this work opens new avenues towards the understanding RVFV-mosquito interactions, and how they impact RVFV emergence and spread.\u003c/p\u003e","manuscriptTitle":"Influence of biotic factors on Rift Valley Fever virus infection dynamics in mosquitoes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 07:15:08","doi":"10.21203/rs.3.rs-9196651/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7ae3298-c567-433e-b82d-fdce24f912bd","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T07:15:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 07:15:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9196651","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9196651","identity":"rs-9196651","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}