Host influence on the eukaryotic virome of sympatric mosquitoes and abundance of diverse viruses with a broad host range.

Mosquitoes harbor a large diversity of eukaryotic viruses. Those viromes probably influence mosquito physiology and the transmission of human pathogens. Nevertheless, their ecology remains largely unstudied. Here, we address two key questions in virome ecology. First, we assessed the influence of mosquito species on virome taxonomic diversity and relative abundance. Contrary to most previous studies, the potential effect of the habitat was explicitly included. Thousands of individuals of Culex poicilipes and Culex tritaeniorhynchus, two vectors of viral diseases, were concomitantly sampled in three habitats over two years. A total of 95 viral taxa from 25 families were identified with meta-transcriptomics, with 75% of taxa shared by both mosquitoes. Viromes significantly differed by mosquito species but not by habitat. Differences were largely due to changes in relative abundance of shared taxa. Then, we studied the diversity of viruses with a broad host range. We searched for viral taxa shared by the two Culex species and Aedes vexans, another disease vector, present in one of the habitats. Twenty-six out of the 163 viral taxa were found in the three mosquitoes. These taxa encompassed 14 families. A database analysis supported broad host ranges for many of those viruses, as well as a widespread geographical distribution. Thus, the viromes of mosquitoes from the same genera mainly differed in the relative abundance of shared taxa, whereas differences in viral diversity dominated between mosquito genera. Whether this new model of virome diversity and structure applies to other mosquito communities remains to be determined.

Effectiveness of a New Self-Marking Technique in Aedes aegypti under Laboratory Conditions.

In the implementation of mosquito control strategy programs using Sterile Insect Technique and other rear and release strategies, knowledge on the dispersion, competitiveness and survival of mosquitos is considered essential. To assess these parameters, marking techniques are generally used to differentiate colony mosquitoes from wild ones. Most of the existing mosquito marking methods require numerous manipulations that can impact their quality. In this study, we have developed a self-marking technique that can reduce the damage associated with mosquito handling. The marking technique consisted of adding fluorescent powder (DayGlo: A-17-N Saturn yellow) directly to the surface water of the receptacle containing Aedes aegypti male pupae. Different quantities of powder were used, and marking efficacy, powder persistence and mosquito survival were assessed. The results show a mean marking rate of 98 ± 1.61%, and the probability of marking increased significantly (p < 0.001) with increasing concentrations of fluorescent powder. Fluorescent powder persisted up to 20 days and did not induce a negative effect on mosquito survival (χ2 = 5.3, df = 7, p = 0.63). In addition, powder transfer did not occur between marked and unmarked populations. This marking method significantly reduces human intervention and mosquito handling during the marking process, improving the quality of marked mosquitoes used to assess SIT programs.

Insight on Bluetongue virus transmission in small ruminants in Senegal.

Bluetongue (BT) is an infectious, arthropod-borne viral disease of domestic and wild ruminants. The disease causes animal mortality, production decrease and commercial limits for herds. Despite the active circulation of the disease in the world, few studies have been carried out in Senegal. The objective of this study was to assess the current prevalence of BT in small ruminants and the serotypes circulating in Senegal. A cross-sectional study was conducted in the fourteen regions of Senegal. After the sampling campaign, sera collected in sheep and goats herds were screened for the presence of Bluetongue virus (BTV) specific antibodies using c-Elisa. The whole blood of seropositive animals was further analyzed by RT-qPCR and positive samples were typed to identify BTV serotypes. Analysis of several risk factors such as age, sex and species of animals was performed using logistic regression. The overall seroprevalence of BTV in Senegal was 72.6% (95% CI: 70.3-74.9%) with 75.9% (95% CI: 72.2-79.5%) in goat and 70.6% (95% CI: 67.5-73.6%) in sheep. Female (prevalence=77.1%) and adult (prevalence=80%) animals showed the highest seropositivity to BTV compared respectively to male (55.7%, p=6.133e-09) and young (49.4%, p < 2.2e-16). The RT-qPCR results showed the presence of BT viral genome in 359 small ruminants. The results obtained from serological and genotyping studies showed an active spread of the Bluetongue virus in domestic ruminants and phylogenetic analysis showed that the BTV-2 is one of the circulating serotypes in Senegal. This study allows having baseline information for controlling Bluetongue in Senegal.

Improving the efficiency of the BG sentinel 2 trap to assess the activity of Aedes (Stegomyia) aegypti [Linnaeus, 1762] in Senegal.

The use of efficient mosquito sampling methods in vector surveillance programs is crucial to inform control actions and prevent outbreaks. amongst existing trapping methods, the BG sentinel trap is widely used for collecting mosquitoes from the subgenus Stegomyia. However, studies state that the BG-sentinel trap underestimates the relative abundance of mosquito vectors. In this study, we used mice to enhance the effectiveness of the BG-sentinel trap to collect Aedes aegypti (Linnaeus) and follow the species' daily abundance under local conditions. The Latin square method was used to compare different combinations in three different seasons. Of the 35,107 mosquitoes collected, Ae. aegypti (53.82%) and Culex quinquefasciatus (46.07%) were dominant. The combination of BG-Lure + 3 mice captured more Ae. aegypti individuals (apparent density per trap/day (ADT = 187.65 ± 133.53; p < 0.001) followed by the 3 mice-baited BG-sentinel trap (ADT = 163.47 ± 117.32), the BG-sentinel trap without attractant (ADT = 74.15 ± 117.07) and the BG-sentinel trap + BG-Lure (ADT = 47.1 ± 115.91). Aedes aegypti showed two peaks of activity in the day, one following the sunrise and one before the sunset, influenced by temperature and relative humidity. Our study suggests the use of mice to enhance the efficiency of the BG-Sentinel trap to catch Ae. aegypti. However, its application in large scale entomological monitoring programs should be difficult because of ethical and operational constraints.

A library preparation optimized for metagenomics of RNA viruses.

Our understanding of the viral communities associated to animals has not yet reached the level attained on the bacteriome. This situation is due to, among others, technical challenges in adapting metagenomics using high-throughput sequencing to the study of RNA viromes in animals. Although important developments have been achieved in most steps of viral metagenomics, there is yet a key step that has received little attention: the library preparation. This situation differs from bacteriome studies in which developments in library preparation have largely contributed to the democratisation of metagenomics. Here, we present a library preparation optimized for metagenomics of RNA viruses from insect vectors of viral diseases. The library design allows a simple PCR-based preparation, such as those routinely used in bacterial metabarcoding, that is adapted to shotgun sequencing as required in viral metagenomics. We first optimized our library preparation using mock viral communities and then validated a full metagenomic approach incorporating our preparation in two pilot studies with field-caught insect vectors; one including a comparison with a published metagenomic protocol. Our approach provided a fold increase in virus-like sequences compared to other studies, and nearly-full genomes from new virus species. Moreover, our results suggested conserved trends in virome composition within a population of a mosquito species. Finally, the sensitivity of our approach was compared to a commercial diagnostic PCR for the detection of an arbovirus in field-caught insect vectors. Our approach could facilitate studies on viral communities from animals and the democratization of metagenomics in community ecology of viruses.

Assessing the Risk of Occurrence of Bluetongue in Senegal.

Bluetongue is a non-contagious viral disease affecting small ruminants and cattle that can cause severe economic losses in the livestock sector. The virus is transmitted by certain species of the genus Culicoides and consequently, understanding their distribution is essential to enable the identification of high-risk transmission areas. In this work we use bioclimatic and environmental variables to predict vector abundance, and estimate spatial variations in the basic reproductive ratio  R0. The resulting estimates were combined with livestock mobility and serological data to assess the risk of Bluetongue outbreaks in Senegal. The results show an increasing abundance of C. imicola, C. oxystoma, C. enderleini, and C. miombo from north to south. R0 < 1 for most areas of Senegal, whilst southern (Casamance) and southeastern (Kedougou and part of Tambacounda) agro-pastoral areas have the highest risk of outbreak (R0 = 2.7 and 2.9, respectively). The next higher risk areas are in the Senegal River Valley (R0 = 1.07), and the Atlantic coast zones. Seroprevalence rates, shown by cELISA, weren't positively correlated with outbreak probability. Future works should include follow-up studies of competent vector abundancies and serological surveys based on the results of the risk analysis conducted here to optimize the national epidemiological surveillance system.

Rift Valley fever in northern Senegal: A modelling approach to analyse the processes underlying virus circulation recurrence.

Rift Valley fever (RVF) is endemic in northern Senegal, a Sahelian area characterized by a temporary pond network that drive both RVF mosquito population dynamics and nomadic herd movements. To investigate the mechanisms that explain RVF recurrent circulation, we modelled a realistic epidemiological system at the pond level integrating vector population dynamics, resident and nomadic ruminant herd population dynamics, and nomadic herd movements recorded in Younoufere area. To calibrate the model, serological surveys were performed in 2015-2016 on both resident and nomadic domestic herds in the same area. Mosquito population dynamics were obtained from a published model trained in the same region. Model comparison techniques were used to compare five different scenarios of virus introduction by nomadic herds associated or not with vertical transmission in Aedes vexans. Our serological results confirmed a long lasting RVF endemicity in resident herds (IgG seroprevalence rate of 15.3%, n = 222), and provided the first estimation of RVF IgG seroprevalence in nomadic herds in West Africa (12.4%, n = 660). Multivariate analysis of serological data suggested an amplification of the transmission cycle during the rainy season with a peak of circulation at the end of that season. The best scenario of virus introduction combined yearly introductions of RVFV from 2008 to 2015 (the study period) by nomadic herds, with a proportion of viraemic individuals predicted to be larger in animals arriving during the 2nd half of the rainy season (3.4%). This result is coherent with the IgM prevalence rate (4%) found in nomadic herds sampled during the 2nd half of the rainy season. Although the existence of a vertical transmission mechanism in Aedes cannot be ruled out, our model demonstrates that nomadic movements are sufficient to account for this endemic circulation in northern Senegal.

Ecological niche modelling to estimate the distribution of Culicoides, potential vectors of bluetongue virus in Senegal.

BACKGROUND: Vector-borne diseases are among the leading causes of morbidity and mortality in humans and animals. In the Afrotropical region, some are transmitted by Culicoides, such as Akabane, bluetongue, epizootic haemorrhagic fever and African horse sickness viruses. Bluetongue virus infection has an enormous impact on ruminant production, due to its high morbidity and mortality rates. METHODS: A nationwide Culicoides trapping campaign was organized at the end of the 2012 rainy season in Senegal. A Maximum Entropy approach (MaxEnt), Boosted Regression Tree (BRT) method and Ecological Niche Factor Analysis (ENFA) were used to develop a predictive spatial model for the distribution of Culicoides, using bio-climatic variables, livestock densities and altitude. RESULTS: The altitude, maximum temperature of the warmest month, precipitation of the warmest quarter, mean temperature of the wettest quarter, temperature seasonality, precipitation of the wettest quarter and livestock density were among the most important factors to predict suitable habitats of Culicoides. Culicoides occurrences were, in most of the cases, positively correlated to precipitation variables and livestock densities; and negatively correlated to the altitude and temperature indices. The Niayes area and the Groundnut basin were the most suitable habitats predicted. CONCLUSION: We present ecological niche models for different Culicoides species, namely C. imicola, C. oxystoma, C. enderleini and C. miombo, potential vectors of bluetongue virus, on a nationwide scale in Senegal. Through our modelling approach, we were able to determine the effect of bioclimatic variables on Culicoides habitats and were able to generate maps for the occurrence of Culicoides species. This information will be helpful in developing risk maps for disease outbreaks.

Host-feeding patterns of Aedes (Aedimorphus) vexans arabiensis, a Rift Valley Fever virus vector in the Ferlo pastoral ecosystem of Senegal.

BACKGROUND: Host-vector contact is a key factor in vectorial capacity assessment and thus the transmission of mosquito-borne viruses such as Rift Valley Fever (RVF), an emerging zoonotic disease of interest in West Africa. The knowledge of the host-feeding patterns of vector species constitutes a key element in the assessment of their epidemiological importance in a given environment. The aim of this work was to identify the blood meal origins of the mosquito Aedes vexans arabiensis, the main vector of RVF virus in the Ferlo pastoral ecosystem of Senegal. METHODOLOGY/PRINCIPAL FINDINGS: Engorged female mosquitoes were collected in Younouféré in the pastoral ecosystem in the Ferlo region during the 2014 rainy season. CO2-baited CDC light traps were set at six points for two consecutive nights every month from July to November. Domestic animals present around traps were identified and counted for each trapping session. Blood meal sources of engorged mosquitoes were identified using a vertebrate-specific multiplexed primer set based on cytochrome b. Blood meal sources were successfully identified for 319 out of 416 blood-fed females (76.68%), of which 163 (51.1%) were single meals, 146 (45.77%) mixed meals from two different hosts and 10 (3.13%) mixed meals from three different hosts. Aedes vexans arabiensis fed preferentially on mammals especially on horse compared to other hosts (FR = 46.83). Proportions of single and mixed meals showed significant temporal and spatial variations according to the availability of the hosts. CONCLUSION: Aedes vexans arabiensis shows an opportunistic feeding behavior depending on the host availability. This species fed preferentially on mammals especially on horses (primary hosts) and ruminants (secondary hosts).

Ecological distribution and population dynamics of Rift Valley fever virus mosquito vectors (Diptera, Culicidae) in Senegal.

BACKGROUND: Many zoonotic infectious diseases have emerged and re-emerged over the last two decades. There has been a significant increase in vector-borne diseases due to climate variations that lead to environmental changes favoring the development and adaptation of vectors. This study was carried out to improve knowledge of the ecology of mosquito vectors involved in the transmission of Rift Valley fever virus (RVFV) in Senegal. METHODS: An entomological survey was conducted in three Senegalese agro-systems, Senegal River Delta (SRD), Senegal River Valley (SRV) and Ferlo, during the rainy season (July to November) of 2014 and 2015. Mosquitoes were trapped using CDC light traps set at ten sites for two consecutive nights during each month of the rainy season, for a total of 200 night-traps. Ecological indices were calculated to characterize the different populations of RVFV mosquito vectors. Generalized linear models with mixed effects were used to assess the influence of climatic conditions on the abundance of RVFV mosquito vectors. RESULTS: A total of 355,408 mosquitoes belonging to 7 genera and 35 species were captured in 200 night-traps. RVFV vectors represented 89.02% of the total, broken down as follows: Ae. vexans arabiensis (31.29%), Cx. poicilipes (0.6%), Cx. tritaeniorhynchus (33.09%) and Ma. uniformis (24.04%). Comparison of meteorological indices (rainfall, temperature, relative humidity), abundances and species diversity indicated that there were no significant differences between SRD and SRV (P = 0.36) while Ferlo showed significant differences with both (P < 0.001). Mosquito collection increased significantly with temperature for Ae. vexans arabiensis (P < 0.001), Cx. tritaeniorhynchus (P = 0.04) and Ma. uniformis (P = 0.01), while Cx. poicilipes decreased (P = 0.003). Relative humidity was positively and significantly associated with the abundances of Ae. vexans arabiensis (P < 0.001), Cx. poicilipes (P = 0.01) and Cx. tritaeniorhynchus (P = 0.007). Rainfall had a positive and significant effect on the abundances of Ae. vexans arabiensis (P = 0.005). The type of biotope (temporary ponds, river or lake) around the trap points had a significant effect on the mosquito abundances (P < 0.001). CONCLUSIONS: In terms of species diversity, the SRD and SRV ecosystems are similar to each other and different from that of Ferlo. Meteorological indices and the type of biotope (river, lake or temporary pond) have significant effects on the abundance of RVFV mosquito vectors.