The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype-dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can have carry-over effects on adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we showed that the bacterial community structure differs between the two genotypes of Ae. aegypti when given identical microbiomes, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific larval microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti. We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Serosurvey of Chikungunya Virus in Old World Fruit Bats, Senegal, 2020-2022.

We conducted a cross-sectional serosurvey for chikungunya virus (CHIKV) exposure in fruit bats in Senegal during 2020-2023. We found that 13.3% (89/671) of bats had CHIKV IgG; highest prevalence was in Eidolon helvum (18.3%, 15/82) and Epomophorus gambianus (13.7%, 63/461) bats. Our results suggest these bats are naturally exposed to CHIKV.

Reemergence of Sylvatic Dengue Virus Serotype 2 in Kedougou, Senegal, 2020.

In 2020, a sylvatic dengue virus serotype 2 infection outbreak resulted in 59 confirmed dengue cases in Kedougou, Senegal, suggesting those strains might not require adaptation to reemerge into urban transmission cycles. Large-scale genomic surveillance and updated molecular diagnostic tools are needed to effectively prevent dengue virus infections in Senegal.

An amplicon-based sequencing approach for Usutu virus characterization.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus.

Concurrent amplification of Zika, chikungunya, and yellow fever virus in a sylvatic focus of arboviruses in Southeastern Senegal, 2015.

BACKGROUND: Chikungunya (CHIKV), yellow fever (YFV) and Zika (ZIKV) viruses circulate in sylvatic transmission cycles in southeastern Senegal, where they share common hosts and vectors. All three viruses undergo periodic amplifications, during which they are detected in mosquitoes and sometimes in hosts. However, little is known about their spatio-temporal patterns in years in which they undergo concurrent amplification. The aim of this study was to describe the co-amplification of ZIKV, CHIKV, and YFV, and the daily dynamics of these arboviruses and theirs vectors within villages in southeastern Senegal. RESULTS: Mosquitoes were collected monthly from July to December 2015. Each evening, from 6 to 9 PM, landing collections were performed by teams of 3 persons working simultaneously in 70 sites situated in forest (canopy and ground), savannah, agriculture, barren, and village (indoor and outdoor) land covers. Collections within villages were continued until 6 AM. Mosquitoes were tested for virus infection by virus isolation and RT-PCR. Seventy-five mosquito pools comprising 10 mosquito species contained at least one virus. Ae. furcifer and Ae. luteocephalus were infected by all three viruses, Ae. taylori by YFV and ZIKV, and remaining seven species by only, only YFV or only ZIKV. No single mosquito pool contained more than one virus. CHIKV was the only virus detected in all land cover classes and was found in the greatest number of sampling sites (32.9%, n = 70). The proportion of sites in which more than one virus was detected was less than 6%. Ae. aegypti formosus, Ae. furcifer, Ae. luteocephalus, Ae. minutus, Ae. vittatus, and An. gambiae were found within villages. These vectors were mainly active around dusk but Ae. furcifer was collected until dawn. All viruses save ZIKV were detected indoors and outdoors, mainly around dusk. Virus positive pools were detected over 2, 3 and 4 months for YFV, CHIKV and ZIKV, respectively. CONCLUSION: Our data indicate that the distribution of different vector species and different arboviruses vary substantially between sites, suggesting that CHIKV, YFV, and ZIKV may have different transmission cycles in Southeastern Senegal.

Zika virus in southeastern Senegal: survival of the vectors and the virus during the dry season.

BACKGROUND: Zika virus (ZIKV, genus Flavivirus, family Flaviviridae) is transmitted mainly by Aedes mosquitoes. This virus has become an emerging concern of global public health with recent epidemics associated to neurological complications in the pacific and America. ZIKV is the most frequently amplified arbovirus in southeastern Senegal. However, this virus and its adult vectors are undetectable during the dry season. The aim of this study was to investigate how ZIKV and its vectors are maintained locally during the dry season. METHODS: Soil, sand, and detritus contained in 1339 potential breeding sites (tree holes, rock holes, fruit husks, discarded containers, used tires) were collected in forest, savannah, barren and village land covers and flooded for eggs hatching. The emerging larvae were reared to adult, identified, and blood fed for F1 production. The F0 and F1 adults were identified and tested for ZIKV by Reverse Transcriptase-Real time Polymerase Chain Reaction. RESULTS: A total of 1016 specimens, including 13 Aedes species, emerged in samples collected in the land covers and breeding sites investigated. Ae. aegypti was the dominant species representing 56.6% of this fauna with a high plasticity. Ae. furcifer and Ae. luteocephalus were found in forest tree holes, Ae. taylori in forest and village tree holes, Ae. vittatus in rock holes. ZIKV was detected from 4 out of the 82 mosquito pools tested. Positive pools included Ae. bromeliae (2 pools), Ae. unilineatus (1 pool), and Ae. vittatus (1 pool), indicating that the virus is maintained in these Aedes eggs during the dry season. CONCLUSION: Our investigation identified breeding sites types and land cover classes where several ZIKV vectors are maintained, and their maintenance rates during the dry season in southeastern Senegal. The maintenance of the virus in these vectors in nature could explain its early amplification at the start of the rainy season in this area.

Potential of selected Senegalese Aedes spp. mosquitoes (Diptera: Culicidae) to transmit Zika virus.

BACKGROUND: Zika virus (ZIKV; genus Flavivirus, family Flaviviridae) is an emerging virus of medical importance maintained in a zoonotic cycle between arboreal Aedes spp. mosquitoes and nonhuman primates in African and Asian forests. Serological evidence and virus isolations have demonstrated widespread distribution of the virus in Senegal. Several mosquito species have been found naturally infected by ZIKV but little is known about their vector competence. METHODS: We assessed the vector competence of Ae. aegypti from Kedougou and Dakar, Ae. unilineatus, Ae. vittatus and Ae. luteocephalus from Kedougou in Senegal for 6 ZIKV strains using experimental oral infection. Fully engorged female mosquitoes were maintained in an environmental chamber set at 27 ± 1 °C and 80 ± 5% Relative humidity. At day 5, 10 and 15 days post infection (dpi), individual mosquito saliva, legs/wings and bodies were tested for the presence of ZIKV genome using real time RT-PCR to estimate the infection, dissemination, and transmission rates. RESULTS: All the species tested were infected by all viral strains but only Ae. vittatus and Ae. luteocephalus were potentially capable of transmitting ZIKV after 15 dpi with 20 and 50% of mosquitoes, respectively, delivering epidemic (HD 78788) and prototype (MR 766) ZIKV strains in saliva. CONCLUSION: All the species tested here were susceptible to oral infection of ZIKV but only a low proportion of Ae. vittatus and Ae. luteocephalus had the viral genome in their saliva and thus the potential to transmit the virus. Further investigations are needed on the vector competence of other species associated with ZIKV for better understanding of the ecology and epidemiology of this virus in Senegal.

Oral susceptibility of Aedes aegypti (Diptera: Culicidae) from Senegal for dengue serotypes 1 and 3 viruses.

OBJECTIVE: To investigate the potential for domestic and wild populations of Aedes aegypti from Dakar and Kedougou to develop a disseminated infection after exposure to DENV-3 and DENV-1. METHODS: We have exposed sylvatic and urban population of Ae. aegypti from Senegal to bloomeals containing dengue serotype 1 and 3. At different incubation period, individual mosquito legs/wings and bodies were tested for virus presence using real time RT-PCR to estimate the infection and dissemination rates. RESULTS: The data indicated low susceptibility to DENV-3 (infection: 2.4-15.2%, and dissemination rates: 0-8.3%) and higher susceptibility to DENV-1 (infection and dissemination rates up to 50%). CONCLUSION: Aedes aegypti from Senegal seem able to develop a disseminated infection of DENV-1 and DENV-3. Further studies are needed to test their ability to transmit the two serotypes.

Dengue 1 outbreak in Rosso, northern Senegal, October 2021: entomologic investigations.

Senegal has experienced periodic epidemics of dengue in urban areas with increased incidence in recent years. However, few data are available on the local ecology of the epidemic vectors. In October 2021, a dengue outbreak was reported in northern Senegal to the Institute Pasteur de Dakar. Entomologic investigations then were undertaken to identify the areas at risk of transmission and to identify the vector(s). Adult mosquitoes were collected indoors and outdoors at selected households, while containers with water were inspected for mosquito larvae. All the Aedes aegypti (L.) collected were tested for dengue virus NS1 protein using a rapid diagnostic test (RDT), and positive samples were confirmed by real-time RT-PCR. The qRT-PCR positive samples were subjected to whole genome sequencing using Nanopore technology. The majority of the larvae-positive containers (83.1%) were used for water storage. The Breteau and Container indices exceeded the WHO-recommended thresholds for the risk of dengue virus transmission except at 2 localities. Ae. aegypti, the only reputed dengue vector, was collected resting indoors as well as outdoors and biting during the day and night. The NS1 protein was detected in 22 mosquito pools, including one pool of females emerging from field-collected larvae. All NS1-positive results were confirmed by RT-PCR. Virus serotyping showed that the outbreak was caused by DENV-1. This study demonstrates the need for continuous control of adult and aquatic stages of Ae. aegypti to prevent future dengue epidemics in Senegal. RDTs appear to be a promising tool for dengue diagnostics and surveillance.

Molecular Detection and Genetic Characterization of Two Dugbe Orthonairovirus Isolates Detected from Ticks in Southern Senegal.

Dugbe virus (DUGV) is a tick-borne arbovirus first isolated in Nigeria in 1964. It has been detected in many African countries using such diverse methods as serological tests, virus isolation, and molecular detection. In Senegal, reports of DUGV isolates mainly occurred in the 1970s and 1980s. Here, we report a contemporary detection of three novel DUGV isolates upon screening of a total of 2877 individual ticks regrouped into 844 pools. The three positive pools were identified as Amblyomma variegatum, the main known vector of DUGV, collected in the southern part of the country (Kolda region). Interestingly, phylogenetic analysis indicates that the newly sequenced isolates are globally related to the previously characterized isolates in West Africa, thus highlighting potentially endemic, unnoticed viral transmission. This study was also an opportunity to develop a rapid and affordable protocol for full-genome sequencing of DUGV using nanopore technology. The results suggest a relatively low mutation rate and relatively conservative evolution of DUGV isolates.

Genomic characterization of a reemerging Chikungunya outbreak in Kedougou, Southeastern Senegal, 2023.

Chikungunya virus has caused millions of cases worldwide over the past 20 years, with recent outbreaks in Kedougou region in the southeastern Senegal, West Africa. Genomic characterization highlights that an ongoing epidemic in Kedougou in 2023 is not due to an introduction event but caused by the re-emergence of an endemic strain evolving linearly in a sylvatic context.

Assessment of the Risk of Exotic Zika Virus Strain Transmission by Aedes aegypti and Culex quinquefasciatus from Senegal Compared to a Native Strain.

Zika virus (ZIKV) shows an enigmatic epidemiological profile in Africa. Despite its frequent detection in mosquitoes, few human cases have been reported. This could be due to the low infectious potential or low virulence of African ZIKV lineages. This study sought to assess the susceptibility of A. aegypti and C. quinquefasciatus to ZIKV strains from Senegal, Brazil, and New Caledonia. Vertical transmission was also investigated. Whole bodies, legs/wings and saliva samples were tested for ZIKV by real-time PCR to estimate infection, dissemination and transmission rates as well as the infection rate in the progeny of infected female A. aegypti. For A. aegypti, the Senegalese strain showed at 15 days post-exposure (dpe) a significantly higher infection rate (52.43%) than the Brazilian (10%) and New Caledonian (0%) strains. The Brazilian and Senegalese strains were disseminated but not detected in saliva. No A. aegypti offspring from females infected with Senegalese and Brazilian ZIKV strains tested positive. No infection was recorded for C. quinquefasciatus. We observed the incompetence of Senegalese A. aegypti to transmit ZIKV and the C. quinquefasciatus were completely refractory. The effect of freezing ZIKV had no significant impact on the vector competence of Aedes aegypti from Senegal, and vertical transmission was not reported in this study.

The influence of the larval microbiome on susceptibility to Zika virus is mosquito genotype dependent.

The microbiome of the mosquito Aedes aegypti is largely determined by the environment and influences mosquito susceptibility for arthropod-borne viruses (arboviruses). Larval interactions with different bacteria can influence adult Ae. aegypti replication of arboviruses, but little is known about the role that mosquito host genetics play in determining how larval-bacterial interactions shape Ae aegypti susceptibility to arboviruses. To address this question, we isolated single bacterial isolates and complex microbiomes from Ae. aegypti larvae from various field sites in Senegal. Either single bacterial isolates or complex microbiomes were added to two different genetic backgrounds of Ae. aegypti in a gnotobiotic larval system. Using 16S amplicon sequencing we show that similarities in bacterial community structures when given identical microbiomes between different genetic backgrounds of Ae. aegypti was dependent on the source microbiome, and the abundance of single bacterial taxa differed between Ae. aegypti genotypes. Using single bacterial isolates or the entire preserved complex microbiome, we tested the ability of specific microbiomes to drive differences in infection rates for Zika virus in different genetic backgrounds of Ae. aegypti . We observed that the proportion of Zika virus-infected adults was dependent on the interaction between the larval microbiome and Ae. aegypti host genetics. By using the larval microbiome as a component of the environment, these results demonstrate that interactions between the Ae. aegypti genotype and its environment can influence Zika virus infection. As Ae. aegypti expands and adapts to new environments under climate change, an understanding of how different genotypes interact with the same environment will be crucial for implementing arbovirus transmission control strategies.

Detection of Crimean-Congo Haemorrhagic Fever Virus from Livestock Ticks in Northern, Central and Southern Senegal in 2021.

Crimean-Congo haemorrhagic fever virus (CCHFV) occurs sporadically in Senegal, with a few human cases each year. This active circulation of CCHFV motivated this study which investigated different localities of Senegal to determine the diversity of tick species, tick infestation rates in livestock and livestock infections with CCHFV. The samples were collected in July 2021 from cattle, sheep and goats in different locations in Senegal. Tick samples were identified and pooled by species and sex for CCHFV detection via RT-PCR. A total of 6135 ticks belonging to 11 species and 4 genera were collected. The genus Hyalomma was the most abundant (54%), followed by Amblyomma (36.54%), Rhipicephalus (8.67%) and Boophilus (0.75%). The prevalence of tick infestation was 92%, 55% and 13% in cattle, sheep and goats, respectively. Crimean-Congo haemorrhagic fever virus (CCHFV) was detected in 54/1956 of the tested pools. The infection rate was higher in ticks collected from sheep (0.42/1000 infected ticks) than those from cattle (0.13/1000), while all ticks collected from goats were negative. This study confirmed the active circulation of CCHFV in ticks in Senegal and highlights their role in the maintenance of CCHFV. It is imperative to take effective measures to control tick infestation in livestock to prevent future CCHFV infections in humans.

Rapid On-Site Detection of Arboviruses by a Direct RT-qPCR Assay.

Arthropod-borne diseases currently constitute a source of major health concerns worldwide. They account for about 50% of global infectious diseases and cause nearly 700,000 deaths every year. Their rapid increase and spread constitute a huge challenge for public health, highlighting the need for early detection during epidemics, to curtail the virus spread, and to enhance outbreak management. Here, we compared a standard quantitative polymerase chain reaction (RT-qPCR) and a direct RT-qPCR assay for the detection of Zika (ZIKV), Chikungunya (CHIKV), and Rift Valley Fever (RVFV) viruses from experimentally infected-mosquitoes. The direct RT-qPCR could be completed within 1.5 h and required 1 µL of viral supernatant from homogenized mosquito body pools. Results showed that the direct RT-qPCR can detect 85.71%, 89%, and 100% of CHIKV, RVFV, and ZIKV samples by direct amplifications compared to the standard method. The use of 1:10 diluted supernatant is suggested for CHIKV and RVFV direct RT-qPCR. Despite a slight drop in sensitivity for direct PCR, our technique is more affordable, less time-consuming, and provides a better option for qualitative field diagnosis during outbreak management. It represents an alternative when extraction and purification steps are not possible because of insufficient sample volume or biosecurity issues.

Spatial and temporal dynamics of West Nile virus between Africa and Europe.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

Human and Livestock Surveillance Revealed the Circulation of Rift Valley Fever Virus in Agnam, Northern Senegal, 2021.

The mosquito-borne disease caused by the Rift Valley Fever Virus (RVFV) is a viral hemorrhagic fever that affects humans and animals. In 1987, RVFV emerged in Mauritania, which caused the first RVFV outbreak in West Africa. This outbreak was shortly followed by reported cases in humans and livestock in Senegal. Animal trade practices with neighboring Mauritania suggest northern regions of Senegal are at high risk for RVF. In this study, we aim to conduct a molecular and serological survey of RVFV in humans and livestock in Agnam (northeastern Senegal) by RT-PCR (reverse transcription real-time polymerase chain reaction) and ELISA (Enzyme-Linked Immunosorbent Assay), respectively. Of the two hundred fifty-five human sera, one (0.39%) tested RVFV IgM positive, while fifty-three (20.78%) tested positive for RVFV IgG. For animal monitoring, out of 30 sheep recorded and sampled over the study period, 20 (66.67%) showed seroconversion to RVFV IgG antibodies, notably during the rainy season. The presence of antibodies increased significantly with age in both groups (p < 0.05), as the force of RVF infection (FOI), increased by 16.05% per year for humans and by 80.4% per month for livestock sheep. This study supports the usefulness of setting up a One Health survey for RVF management.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

Mosquito vector competence for dengue is modulated by insect-specific viruses.

Aedes aegypti and A. albopictus mosquitoes are the main vectors for dengue virus (DENV) and other arboviruses, including Zika virus (ZIKV). Understanding the factors that affect transmission of arboviruses from mosquitoes to humans is a priority because it could inform public health and targeted interventions. Reasoning that interactions among viruses in the vector insect might affect transmission, we analysed the viromes of 815 urban Aedes mosquitoes collected from 12 countries worldwide. Two mosquito-specific viruses, Phasi Charoen-like virus (PCLV) and Humaita Tubiacanga virus (HTV), were the most abundant in A. aegypti worldwide. Spatiotemporal analyses of virus circulation in an endemic urban area revealed a 200% increase in chances of having DENV in wild A. aegypti mosquitoes when both HTV and PCLV were present. Using a mouse model in the laboratory, we showed that the presence of HTV and PCLV increased the ability of mosquitoes to transmit DENV and ZIKV to a vertebrate host. By transcriptomic analysis, we found that in DENV-infected mosquitoes, HTV and PCLV block the downregulation of histone H4, which we identify as an important proviral host factor in vivo.

Sublingual misoprostol versus standard surgical care for treatment of incomplete abortion in five sub-Saharan African countries.

BACKGROUND: In low-resource settings, where abortion is highly restricted and self-induced abortions are common, access to post-abortion care (PAC) services, especially treatment of incomplete terminations, is a priority. Standard post-abortion care has involved surgical intervention but can be hard to access in these areas. Misoprostol provides an alternative to surgical intervention that could increase access to abortion care. We sought to gather additional evidence regarding the efficacy of 400 mcg of sublingual misoprostol vs. standard surgical care for treatment of incomplete abortion in the environments where need for economical non-surgical treatments may be most useful. METHODS: A total of 860 women received either sublingual misoprostol or standard surgical care for treatment of incomplete abortion in a multi-site randomized trial. Women with confirmed incomplete abortion, defined as past or present history of vaginal bleeding during pregnancy and an open cervical os, were eligible to participate. Participants returned for follow-up one week later to confirm clinical status. If abortion was incomplete at that time, women were offered an additional follow-up visit or immediate surgical evacuation. RESULTS: Both misoprostol and surgical evacuation are highly effective treatments for incomplete abortion (misoprostol: 94.4%, surgical: 100.0%). Misoprostol treatment resulted in a somewhat lower chance of success than standard surgical practice (RR = 0.90; 95% CI: 0.89-0.92). Both tolerability of side effects and women's satisfaction were similar in the two study arms. CONCLUSION: Misoprostol, much easier to provide than surgery in low-resource environments, can be used safely, successfully, and satisfactorily for treatment of incomplete abortion. Focus should shift to program implementation, including task-shifting the provision of post-abortion care to mid- and low- level providers, training and assurance of drug availability. TRIAL REGISTRATION: This study has been registered at clinicaltrials.gov as NCT00466999 and NCT01539408.