Mosquito-Based Detection of Endogenous Jaagsiekte Sheep Retrovirus in Senegal: Expanding the Scope of Xenosurveillance

Mosquito-Based Detection of Endogenous Jaagsiekte Sheep Retrovirus in Senegal: Expanding the Scope of Xenosurveillance | 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 Mosquito-Based Detection of Endogenous Jaagsiekte Sheep Retrovirus in Senegal: Expanding the Scope of Xenosurveillance Marie Henriette Dior Ndione, El Hadji Ndiaye, Madeleine Dieng, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5951454/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jun, 2025 Read the published version in One Health Outlook → Version 1 posted 10 You are reading this latest preprint version Abstract Background Mosquitoes are well-known vectors for arthropod-borne viruses, yet their role as passive carriers of non-arthropod-borne viruses remains underexplored. Xenosurveillance, a method that utilizes blood-feeding arthropods to sample host and pathogen genetic material, has emerged as a valuable tool in viral ecology. In this study, we report the first identification of Jaagsiekte Sheep Retrovirus (JSRV)-related sequences in blood-fed mosquitoes collected in Senegal. JSRV, a betaretrovirus responsible for ovine pulmonary adenocarcinoma, is typically found in sheep, but its genetic trace in mosquitoes offers a novel perspective on host–vector contact and surveillance. Our study aimed to investigate whether mosquitoes can serve as sentinels for detecting both pathogens and host-derived markers in complex ecosystems. Methods Mosquitoes were collected between 2016 and 2019 from three ecologically significant regions in Senegal (Louga, Barkedji, and Kedougou). Blood-fed mosquitoes were pooled and subjected to RNA extraction and metagenomic sequencing using Illumina NextSeq550. Sequencing data were analyzed with CZ-ID and BLAST for viral identification. RT-qPCR assays were designed to validate the presence of JSRV-related sequences, targeting conserved regions of the envelope gene and 3' untranslated region. Phylogenetic analysis was conducted using MAFFT and IQ-TREE to compare the detected sequence with global exogenous and endogenous JSRV references. Results A diverse array of viruses across mosquito species, including both arboviruses and non-arthropod-borne viruses. A JSRV-related sequence was detected in a single blood-fed mosquito pool collected in Barkedji (2019). The RT-qPCR assay confirmed JSRV presence, validating the sequencing results. Phylogenetic analysis revealed strong similarity to known endogenous JSRV (enJSRV) sequences integrated in the sheep genome, indicating that the detected material likely originated from host DNA ingested during blood feeding. Discussion This study presents the first report of endogenous retroviral sequences detected in mosquitoes, alongside the identification of actively circulating viruses, highlighting the broader potential of mosquitoes as environmental sentinels. While mosquitoes are not biological vectors for JSRV, their ability to capture both host-derived retroviral material and pathogenic viral genomes through bloodmeals reinforces the value of xenosurveillance for monitoring livestock–vector–environment interactions. These findings contribute to broader efforts in integrated disease surveillance and underscore the utility of combining metagenomics with molecular diagnostics to detect diverse viral signals in high-risk ecological settings. Jaagsiekte sheep Retrovirus xenosurveillance mosquitoes Senegal metagenomics virus–host–vector interactions Figures Figure 1 Background The discovery of viruses transmitted by arthropod vectors has transformed our understanding of viral ecology and infectious disease dynamics [ 1 ]. Mosquitoes, in particular, play a critical role in transmitting a wide range of pathogens that affect both animal and human populations [ 2 ]. This fact has prompted extensive research into the complex interactions between vectors, viruses, and hosts, with a special focus on zoonotic spillover events where pathogens cross species barriers [ 3 ]. Advances in next-generation sequencing (NGS) technologies have further accelerated pathogen discovery, especially in the context of vector-borne diseases. NGS enables high-throughput, unbiased sequencing, allowing for the detection of both known and novel viruses in diverse ecological settings [ 4 ]. Xenosurveillance, or the sampling of pathogen genetic material from the blood meals of arthropod vectors, has proven effective for detecting viruses circulating in wildlife and domestic animals [ 5 , 6 ]. When combined with metagenomic sequencing, xenosurveillance offers comprehensive insights into viral diversity in specific environments. This approach not only aids in the discovery of new pathogens but also helps clarify viral transmission ecology, identifying potential vectors and reservoirs for zoonotic diseases [ 7 ]. Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus known to cause ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer in sheep [ 8 ]. Transmission occurs primarily via aerosolized droplets, and the virus has been considered tightly restricted to sheep and closely related ruminants. Two forms of JSRV exist: the exogenous, infectious virus (exJSRV), and multiple endogenous variants (enJSRVs), which are stably integrated into the host genome. These endogenous forms share high sequence similarity with exJSRV and retain some functional elements, raising interest in their ecological relevance and evolutionary relationship. Traditionally, arthropods have not been implicated in JSRV transmission, and no vector-borne involvement has been demonstrated. In this study, alongside other arthropod-borne and non-arthropod-borne viruses, we report the first detection of JSRV-related genetic material in blood-fed mosquitoes collected in southeastern Senegal, identified through a vector-enabled metagenomic approach. Phylogenetic analysis revealed that the sequence is most closely related to enJSRV. While this finding does not indicate active viral replication or vector-borne transmission, it provides indirect evidence of mosquito contact with local ruminant hosts, particularly sheep. This supports the growing utility of xenosurveillance for capturing host-derived genetic signatures, in addition to circulating pathogens. Moreover, it aligns with emerging ecological observations, such as the recent detection of JSRV RNA in surface water from Tanzanian waterholes during dry seasons [ 9 ], which together raise important questions about non-traditional interfaces of viral exposure and environmental persistence in pastoral ecosystems. By capturing host-derived retroviral sequences through mosquito metagenomics, our work underscores the broader potential of xenosurveillance to advance understanding of host–vector–environment interactions and to support integrated pathogen and exposure monitoring systems at the intersection of animal and public health. Methods Study sites Blood-fed mosquitoes were collected between 2016 and 2019 from three ecologically significant regions in Senegal, Louga in the northwest, Barkedji in the central Ferlo region, and Kedougou in the southeast, each chosen for their relevance to vector-borne disease dynamics. Louga serves as a transitional zone between agriculture and pastoralism, where fluctuating rainfall and rising temperatures impact livestock and crop production, fostering mosquito-host interactions [ 10 ]. Barkedji, centrally located, features pastoral corridors and wildlife reserves, providing a critical interface for zoonotic spillovers [ 11 ]. Pastoral activities in the region are marked by seasonal livestock movements, with sheep and goats forming the backbone of herds due to their resilience to water scarcity. Kedougou lies within a tropical forest zone near the Guinean border, where the humid climate supports high mosquito activity, making it a hotspot for pathogen circulation [ 12 , 13 ]. Although pastoralism is less dominant in this area compared to the Ferlo or Louga regions, Kedougou has become an important destination for herders from northern Senegal due to its abundant pasture and water resources, especially during dry seasons. Herders are drawn to Kedougou for economic opportunities, including livestock markets where animals command attractive prices, enabling them to diversify their income sources [ 14 ]. Mosquito collection and sample selection This study analyzed archived mosquito pools collected biweekly between 2016 and 2019 as part of the national arbovirus surveillance program coordinated by IPD. Mosquitoes were collected using a variety of trapping methods described by Ndiaye et al., including CDC light traps baited with CO₂, placed at both ground and canopy levels [ 15 ]. Traps were deployed near livestock enclosures, village edges, and natural water sources in ecologically diverse regions such as Barkedji, Louga, and Kedougou. Following morphological identification on the field, mosquito pools were archived at the Centre de Recherche sur les Arbovirus (CRORA) biobank [ 16 ]. For this study, a subset of pools was pre-selected based on their ability to induce cytopathic effect (CPE) upon inoculation in mosquito and mammal cell culture but had tested negative by indirect immunofluorescence assay (IFA) for common arboviruses [ 17 ]. This selection strategy prioritized samples likely to contain uncharacterized or non-target viral agents. Whole mosquito specimens from the selected pools were homogenized, and total nucleic acids were extracted for metagenomic sequencing to identify both host- and pathogen-derived viral material. RNA Extraction and Metagenomic Sequencing RNA extraction was performed using the MagMax Viral/Pathogen Nucleic Acid Isolation Kit and the KingFisher Flex Purification System (Thermo Fisher), following the manufacturer’s recommendations. Sequencing was conducted on the extracted RNA using an Illumina-based unbiased approach, as described previously [ 18 ]. Briefly, host ribosomal RNA was depleted using specific rRNA-targeting probes [ 19 ] and RNase H, and the remaining RNA served as the template for first-strand cDNA synthesis with the SuperScript IV Reverse Transcriptase kit. Double-stranded cDNA was then generated using Klenow fragment DNA polymerase. Libraries were prepared with the Nextera XT DNA Library Preparation kit (Illumina), and sequencing was carried out on the NextSeq 550 system. The resulting sequences were analyzed using the CZ-ID metagenomics platform ( http://czid.org , accessed on 15 March 2023) for viral identification [ 20 ]. Default pipeline parameters were used, incorporating quality control steps such as adapter trimming, low-complexity read filtering, and the removal of host-derived reads (sheep, goat, human) as well as common laboratory contaminants. Sequence alignment was performed against curated nucleotide (NT) and protein (NR) databases using Bowtie2 and RAPSearch2, respectively. Reads were assembled into contigs with SPAdes, and taxonomic classification was based on nucleotide and translated alignments, applying identity thresholds of approximately 80–85% for NT and 90–95% for NR, with e-value cutoffs set at ≤ 1e-10. Additionally, the assembled sequences were subjected to BLAST analysis (Basic Local Alignment Search Tool) against the NCBI nucleotide database to confirm viral identity and assess sequence similarity with known viral genomes, facilitating more precise taxonomic classification and strain identification. Molecular Assay Development To confirm the presence of JSRV detected through NGS, we developed two TaqMan-based reverse transcription quantitative PCR (RT-qPCR) assay were developed. These assays utilized primers and a fluorescently labeled probe designed with Primer3 [ 21 ], specifically targeting regions of the envelope gene and the 3' untranslated region (3’UTR) based on our assembled JSRV sequences. The designed oligonucleotide sets were rigorously evaluated in silico for specificity using BLAST analysis, ensuring that they would exclusively bind to the target JSRV sequences without cross-reactivity with other viral or host genomes. In addition to the in-house RT-qPCR, we employed the hemi-nested U3hn PCR, a previously described method targeting the U3 region of the viral long terminal region (LTR), and known for its high sensitivity in field studies [ 22 ]. This assay was used to complement and validate the results obtained with our newly developed RT-qPCR assay. All assays were performed on RNA extracted from mosquito pools, providing a robust confirmation framework for the detection of JSRV-related sequences. Jaagsiekte virus Phylogenetic Analysis The consensus data generated during this study were aligned with a representative dataset of available sequences using the multiple sequence alignment tool MAFFT (version 7) [ 23 ] with default parameters. This alignment allowed for a comprehensive comparison of the newly detected sequence with known viral genomes, providing a robust framework to establish their evolutionary relationships. The aligned sequences were then used to construct a maximum likelihood (ML) phylogenetic tree, employing IQ-TREE [ 24 ], a widely recognized tool for phylogenetic inference. The best-fit substitution model for the analysis was selected using ModelFinder [ 25 ], ensuring an accurate representation of the evolutionary processes underlying the data. The phylogenetic tree was visualized using FigTree (version 1.4.4) ( http://tree.bio.ed.ac.uk/software/figtree/ , accessed on 17 March 2023), a flexible software for exploring and presenting phylogenetic results. Results Viral Diversity in Mosquito Populations, Co-detection Patterns and Regional Variation A total of 78 mosquito pools collected between 2016 and 2019 were included in the metagenomic analysis. Although our primary focus was on blood-fed female mosquitoes, some pools also included non-blood-fed females and a few males due to the retrospective nature of the archived material. These pools were derived from diverse mosquito species and sites across Senegal. Our metagenomic sequencing identified a wide array of viruses across various mosquito species collected from different regions of Senegal between 2016 and 2019 (Table 1 ). These included both arboviruses and non-arboviruses, demonstrating the remarkable diversity of viral communities harbored by mosquitoes. Notable detections were observed in species such as Aedes aegypti , Aedes vexans , Culex quinquefasciatus , and Aedes furcifer , each playing a distinct role in the viral ecosystem. The identified viruses spanned multiple viral species, with significant detections of Pestivirus A, Dezidougou virus, and Culex iflavivirus, reflecting a broad spectrum of viral types circulating in the environment. Co-detections were frequently observed across several mosquito species, suggesting complex viral interactions within vector populations. While multiple viruses were identified in some pooled samples, we refer to these as instances of co-detection rather than confirmed co-infections. Given that each pool may contain multiple mosquitoes, the detected viral sequences could originate from different individuals. As such, these findings reflect aggregate viral presence within the pool and do not indicate concurrent infection of a single mosquito. The metrics associated with viral identification are provided in supplementary table 1 . Table 1 Mosquito Species and Viruses Identified Across Collection Sites (2016–2019) Mosquito species Collection locality Sex Date of Collection (Year-Month) Identified Viruses Number of pools Number of individuals Feeding Status Aedes aegypti Louga Female 2017-10 - 2 2 Fed-blood Male Aedes Albopictus Densovirus / Dezidougou virus 1 1 Unfed 2017-11 Dezidougou virus 1 2 Unfed Pestivirus A 1 1 Unfed Kedougou Female 2019-10 Pestivirus A 1 2 Unfed Pestivirus A / Taesano Aedes Virus 1 2 Fed-blood Taesano Aedes Virus 1 4 Unfed Aedes centropunctatus Kedougou Female 2019-10 Pestivirus A 1 1 Unfed Aedes dalzieli Barkedji Female 2017-08 - 1 5 Unfed 2019-09 - 1 1 Unfed Kedougou 2019-10 Pestivirus A 2 58 Unfed Aedes furcifer Kedougou Female 2019-10 Pestivirus A /Mosquito Densovirus BR/07 1 2 Fed-blood Aedes ochraceus Barkedji Female 2017-08 - 1 5 Unfed Aedes sudanensis Barkedji Female 2017-08 - 1 1 Unfed 2019-09 Dezidougou Virus / Daezongdong Virus 1 1 Unfed Aedes taylori Kedougou Female 2019-10 - 1 1 Fed-blood Aedes unilineatus Barkedji Female 2019-09 Jaagsiekte Sheep Retrovirus / Dezidougou Virus 1 3 Fed-blood Pestivirus A 1 2 Fed-blood Aedes vexans Barkedji Female 2016-09 - 6 213 Unfed 2017-08 - 4 114 Unfed Dezidougou virus / Aedes vexans iflavivirus 1 40 Unfed 2019-08 - 1 50 Unfed Dezidougou Virus 4 200 Unfed 2019-09 - 1 1 Unfed Aedes Vexans iflavivirus 1 3 Unfed Aedes Vexans iflavivirus / Dezidougou Virus 1 6 Unfed Dezidougou Virus 3 71 Fed-blood Anopheles pharaonsis Barkedji Female 2017-08 - 1 3 Unfed Male - 1 1 Unfed Culex quinquefasciatus Louga Female 2017-10 Culex iflavivirus / Pestivirus H 1 5 Fed-blood Hubei partiti-like Virus 1 1 Unfed Hubei partiti-like Virus / Avian Leukosis Virus / Yongsan iflavivirus/Culex iflavivirus 1 2 Fed-blood 2017-11 - 4 66 Fed-blood Culex iflavivirus / Avian leukosis Virus 1 39 Fed-blood Male 2017-11 - 1 35 Unfed Aedes Albopictus Densovirus 1 5 Unfed Culex flavivirus /Culex Densovirus/ Mosquito Densovirus BR/07 1 16 Unfed Pestivirus H / Hubei mosquito virus / Culex iflavivirus 1 15 Unfed Culex tritaeniorhyncus Louga Female 2017-11 - 2 2 Unfed Culex antennatus Barkedji Female 2017-08 - 1 1 Unfed Culex ethiopicus Barkedji Female 2016-09 - 1 11 - 2019-09 - 1 25 - Pestivirus A / Dezidougou Virus 1 37 - Culex neavei Barkedji Female 2019-10 Barkedji Virus / Bagaza Virus / Sindbis Virus / West-Nile Virus 1 50 - Barkedji Virus / Bagaza Virus / Usutu Virus 1 50 - Culex perfuscus Barkedji Female 2016-09 - 1 4 - Culex poicilipes Barkedji Female 2017-08 - 2 3 - - 1 6 - 2019-09 - 1 33 - Dezidougou Virus / Aedes vexans iflavivirus 1 50 - Dezidougou Virus / Daezongdong Virus 1 50 - Pestivirus A 2 37 Fed-blood Culex quinquefasciatus Barkedji Female 2019-09 Aedes Pseudocutellaris Reovirus / Pestivirus A 1 1 Fed-blood Dezidougou Virus 1 1 - Pestivirus A 1 1 Fed-blood Culex tigripes Barkedji Female 2017-08 - 1 1 - Phlebotom sp Barkedji Female 2019-10 - 1 4 - For example, Pestivirus A, known as bovine viral diarrhea virus type 1 infecting various vertebrates including cattle, sheep, goats, pigs and wild ruminants was widely detected in mosquitoes from multiple regions, including Kedougou and Barkedji, particularly in species like Aedes unilineatus and Aedes furcifer . Dezidougou virus, another frequently identified virus, was found alongside Aedes vexans iflavivirus in Aedes vexans pools from Barkedji. In Culex quinquefasciatus from Louga, co-detections were observed, with Culex iflavivirus and Pestivirus H appearing in the same pools as Hubei partiti-like virus and Avian Leukosis virus, highlighting the complexity of viral ecosystems in the region. These findings underscore the extensive viral diversity present within mosquito populations, with marked regional variation observed across the study sites. Distinct viral profiles were found in Barkedji (51 pools, 1,085 mosquitoes), Louga (19 pools, 192 mosquitoes), and Kedougou (8 pools, 70 mosquitoes), likely influenced by local ecological factors. Barkedji and Louga each harbored 13 unique viral species, while Kedougou, a tropical forest site, had only 3. This may reflect our sampling strategy, which focused on pools that induced cytopathic effects in mosquito or mammalian cell cultures but tested negative for common arboviruses, enhancing the detection of uncharacterized or non-target viruses. Such variation points to the critical role of regional climate, host availability, and habitat conditions in shaping mosquito-associated viral communities. Notably, co-detections within the same mosquito pools were more common in samples from Louga and Barkedji, suggesting that these regions may be hotspots for viral transmission and co-circulation. While we cannot infer true co-infections at the individual mosquito level due to the use of pooled samples, these patterns may reflect overlapping ecological niches or shared host exposure, facilitating the simultaneous presence of multiple viral species in the same sampling units. Detection of Jaagsiekte Sheep Retrovirus Among the various viruses identified, our analysis revealed the first detection of JSRV in mosquitoes. From a blood-fed mosquito pool collected in Barkedji in 2019, we recovered a partial genome sequence (about 600 base pairs in length) encompassing the terminal region of the Env gene and 3'UTR of JSRV. Based on these findings, we developed two sets of molecular assays to confirm the taxonomic identity of JSRV (Table 2 ). Table 2 Jaagsiekte sheep retrovirus-targeted RT-qPCR assays designed during the study. RT-PCR set 1 Enveloppe gene JaagsiekteV_Left primer_1 CGGTTCTGACTGTTGTGCTT JaagsiekteV_Right primer_1 CGCAGCTCCCCTCTCTTTAT JaagsiekteV_Probe_1 FAM - AACATGTTGCAACACCGACA - BBQ RT-PCR set 2 3' untranslated region JaagsiekteV_Left primer_2 CCTAAGCTCCCTGTCCCG JaagsiekteV_Right primer_2 GCCTTCCTTTATTGTGCTGC JaagsiekteV_Probe_2 FAM - TGTGAATGTCAGAAGTCACGT - BBQ To further support these results, we also employed the previously validated U3hn PCR to complement our in-house assays. The U3hn PCR served as an independent molecular confirmation of JSRV presence, particularly useful in low-viral-load samples. RT-qPCR and U3hn PCR were both performed on the mosquito pool in which JSRV was initially detected, along with all nine additionally blood-fed pools with no prior evidence of infection. The assays confirmed JSRV presence in the original mosquito pool (Supplementary table 2 ), though follow-up sequencing did not yield additional coverage of the viral genome. Phylogenetic Analysis of Jaagsiekte Sheep Retrovirus To further characterize the JSRV-related sequence detected in mosquitoes, we performed a phylogenetic analysis using a multiple sequence alignment covering a region of the partial Env gene and the LTR. This alignment included reference betaretroviruses, with particular emphasis on both exJSRV and endogenous (enJSRV) JSRV sequences. A ML phylogenetic tree was then constructed to determine the evolutionary placement of the mosquito-derived sequence. The resulting tree (Fig. 1 ) revealed that the sequence clusters closely with enJSRV strains integrated in the sheep genome, rather than with known exogenous, infectious variants (exJSRV). High bootstrap support (> 90%) on key branches further confirms the robustness of these evolutionary relationships. Discussion This study provides significant insights into the viral ecosystems within mosquito populations in Senegal, identifying a diverse array of viruses, including Pestivirus A, Dezidougou virus, and Culex iflavivirus. The extensive detection of these viruses across various mosquito species, underscores the complexity of viral interactions within vector populations. In the context of xenosurveillance, platforms like CZ-ID provide a standardized approach for detecting viral sequences in blood-fed mosquitoes. However, reliance on default parameters may reduce sensitivity to highly divergent or novel viruses, particularly in ecologically rich settings, potentially leading to an underestimation of viral diversity, a limitation we acknowledge in our interpretation. These findings highlight the critical role that mosquitoes play, not only as vectors of well-established arboviruses but also as reservoirs or passive carriers of non-arboviral pathogens [ 6 ]. This emphasizes the need for broader surveillance efforts to understand the contributions of mosquito populations to viral ecology and pathogen transmission. The detection of JSRV-related genetic material in mosquitoes through unbiased metagenomic sequencing adds a novel dimension to vector-based surveillance. Phylogenetic analysis indicates that the detected sequence is most closely related to enJSRV, non-infectious retroviral elements integrated into the sheep genome. While this does not suggest active viral replication or vector-borne transmission, it provides indirect molecular evidence of mosquito feeding on local ruminant hosts, particularly plausible in Barkedji, a pastoral region characterized by seasonal livestock movements and a high density of sheep and goats. This finding highlights the potential of mosquitoes to act as biological samplers, capturing host-derived viral sequences through bloodmeals, and supports the application of xenosurveillance for ecological monitoring of livestock exposure. The detection of JSRV-related sequences in mosquitoes also aligns with prior findings from Nigeria and Cameroon, where JSRV-related sequences were identified in human samples [ 26 ]. While the biological relevance of those findings remains uncertain, they collectively raise important questions about the distribution and detectability of endogenous retroviral elements across species and environments [ 27 ]. In our study, the identification of enJSRV-like sequences in mosquito bloodmeals, alongside the detection of other viruses such as Pestivirus A, Dezidougou virus, and Culex iflavivirus, highlights the value of combining xenosurveillance and metagenomic tools to monitor both actively circulating viruses and genomic material derived from host organisms. This approach provides a window into local livestock exposure and virus–host–vector interactions without the need for direct sampling of animals. Further investigation is warranted to better understand the ecological roles of retroviruses like enJSRV and their incidental detection in non-host organisms such as arthropod vectors [ 28 ]. Previous studies in Senegal have reported the presence of ovine betaretroviruses, such as ENTV, directly in infected tissues [ 29 ]. By expanding this lens to vector-based environmental surveillance, our findings underscore the potential of mosquitoes to act as passive carriers and ecological sentinels, capturing signals of both active infections and host genomic signatures that may otherwise go undetected. The utility of xenosurveillance as a tool for pathogen detection is further exemplified in this study. By analyzing viral genetic material obtained from blood-feeding mosquitoes, we were able to identify viruses circulating within local wildlife and domestic animal populations without the need for direct mammalian sampling. This method offers a less invasive and more practical approach to surveillance, particularly in remote or logistically challenging regions. Xenosurveillance not only expands our understanding of viral geographic distribution and host interactions but also enhances the early detection of emerging zoonotic threats [ 30 ]. This approach is especially relevant in areas with high biodiversity and frequent interactions between wildlife, domestic animals, and humans, such as Senegal. Beyond its contributions to pathogen discovery, this study emphasizes the critical role of molecular diagnostics in enhancing viral surveillance. The development of specific molecular assays targeting JSRV-related sequences, informed by NGS data, enabled us to confirm the presence of endogenous retroviral material in mosquito samples. This integration of high-throughput sequencing with targeted molecular tools exemplifies a robust approach not only for pathogen detection but also for identifying host-derived genetic signatures within vector populations. It also underscores the importance of adapting diagnostic approaches to capture both arboviral and non-arboviral elements, thereby expanding the scope of mosquito-based surveillance beyond classical vector-borne disease paradigms [ 31 ]. The implications of this study extend to public health preparedness, as the early detection of emerging viral threats can inform targeted interventions and outbreak prevention strategies. Ultimately, this work highlights the transformative potential of integrating xenosurveillance with next-generation sequencing and molecular diagnostics to enhance our understanding of virus-vector-host interactions. By detecting host-derived endogenous retroviral sequences in mosquito bloodmeals, xenosurveillance can provide insight into both pathogen presence and host exposure, capturing ecological contact patterns that extend beyond classical infection-based surveillance. This approach offers a scalable and minimally invasive tool for monitoring livestock health and supporting early-warning systems at the intersection of animal and public health, particularly in pastoral settings where direct sampling remains a challenge. As global health systems move toward integrated One Health frameworks, mosquito-based xenosurveillance offers a unique opportunity to strengthen disease surveillance across species and environments. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated or analyzed in this study are provided within the article and its supplementary materials, while the genomic data are publicly accessible in the NCBI repository. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the NIH West African Center for Emerging Infectious Diseases [grant number U01AI151801-01], the NIH PICREID [grant number U01AI151758] and the Africa CDC Pathogen Genomics Initiative funds [CARES grant 4306-22-EIPHLSSGENOMICS]. Authors' contributions MHDN, MD, SS, MK, MM, SMBSD, SFMD and MMD contributed to the laboratory experiments; EHN, BD, DD, NMS, FAS and AG contributed to the field sampling and species identification; MHDN, AG and MMD designed the study; MHDN, EHN, AG and MMD contributed to the data analysis and curation; AAS, OF, ND, SCW, OumF, MawD, GF and MMD provided administrative and logistical support; MHDN, EHN, AG and MMD drafted the manuscript; all authors reviewed and approved the final version of the manuscript. Acknowledgements We thank the technical staff of the Institut Pasteur de Dakar for their invaluable support in during this work. Their expertise and dedication were essential to the success of this study. References Cuthbert RN, et al. Invasive hematophagous arthropods and associated diseases in a changing world. Parasites Vectors. 2023;16:291. 10.1186/s13071-023-05887-x . Perveen N, et al. Host-pathogen interaction in arthropod vectors: Lessons from viral infections. 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Xenosurveillance: a novel mosquito-based approach for examining the human-pathogen landscape. PLoS Negl Trop Dis. 2015;9(3):e0003628. 10.1371/journal.pntd.0003628 . Huang YS, et al. Arbovirus-Mosquito Vector-Host Interactions and the Impact on Transmission and Disease Pathogenesis of Arboviruses. Front Microbiol. 2019;10:22. 10.3389/fmicb.2019.00022 . Additional Declarations No competing interests reported. Supplementary Files NdioneetalXenosuveillanceSupplementaryTable1OneHealthOutlookID031e2a71e5364a5d9f2ddad3d1b997bd.xlsx NdioneetalXenosuveillanceSupplementaryTable2OneHealthOutlookID031e2a71e5364a5d9f2ddad3d1b997bd.docx Cite Share Download PDF Status: Published Journal Publication published 10 Jun, 2025 Read the published version in One Health Outlook → Version 1 posted Editorial decision: Revision requested 23 May, 2025 Reviews received at journal 23 May, 2025 Reviews received at journal 01 May, 2025 Reviewers agreed at journal 01 May, 2025 Reviews received at journal 28 Apr, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviewers invited by journal 24 Apr, 2025 Submission checks completed at journal 16 Apr, 2025 First submitted to journal 12 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5951454","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447428630,"identity":"a479731f-7656-4fba-a52b-18edc79047fd","order_by":0,"name":"Marie Henriette Dior Ndione","email":"","orcid":"","institution":"Institut Pasteur de Dakar","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"Henriette Dior","lastName":"Ndione","suffix":""},{"id":447428631,"identity":"d376ba9b-5aa4-4068-9f5f-17fe6a5aa0c5","order_by":1,"name":"El Hadji Ndiaye","email":"","orcid":"","institution":"Institut Pasteur de 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Souna","lastName":"Diop","suffix":""},{"id":447428645,"identity":"8be8f464-6411-41dc-940f-45c45febb5d8","order_by":11,"name":"Serge Freddy Moukaha Doukanda","email":"","orcid":"","institution":"Institut Pasteur de Dakar","correspondingAuthor":false,"prefix":"","firstName":"Serge","middleName":"Freddy Moukaha","lastName":"Doukanda","suffix":""},{"id":447428646,"identity":"57506a6f-873d-438f-9873-0d4d8ff2c126","order_by":12,"name":"Amadou Alpha Sall","email":"","orcid":"","institution":"Institut Pasteur de Dakar","correspondingAuthor":false,"prefix":"","firstName":"Amadou","middleName":"Alpha","lastName":"Sall","suffix":""},{"id":447428647,"identity":"d7ccc362-2f50-4172-9000-457b05b64d26","order_by":13,"name":"Ousmane Faye","email":"","orcid":"","institution":"Institut Pasteur de Dakar","correspondingAuthor":false,"prefix":"","firstName":"Ousmane","middleName":"","lastName":"Faye","suffix":""},{"id":447428648,"identity":"3acd71d5-1797-4579-b732-a5b58e3c2f7c","order_by":14,"name":"Ndongo 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Diagne","email":"data:image/png;base64,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","orcid":"","institution":"Institut Pasteur de Dakar","correspondingAuthor":true,"prefix":"","firstName":"Moussa","middleName":"Moise","lastName":"Diagne","suffix":""}],"badges":[],"createdAt":"2025-02-03 13:38:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5951454/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5951454/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s42522-025-00155-7","type":"published","date":"2025-06-10T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81410886,"identity":"46162f8c-a139-45e1-b3e2-f9cf90b6a9d7","added_by":"auto","created_at":"2025-04-25 20:29:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of Jaagsiekte Sheep Retrovirus (JSRV)\u003c/strong\u003e-related sequence detected \u003cstrong\u003ein mosquitoes from Senegal.\u003c/strong\u003e Maximum likelihood tree constructed from partial Env–LTR sequences, including the mosquito-derived sequence and representative \u003cstrong\u003eexogenous (exJSRV, shown with purple branches), endogenous (enJSRV, shown with green branches), and outgroup\u003c/strong\u003e betaretroviruses (shown with black branches). The Senegalese JSRV sequence clusters closely with known \u003cstrong\u003eendogenous JSRVs\u003c/strong\u003e, supporting its origin from host genomic material rather than an exogenous infectious strain. Bootstrap values are displayed for key nodes to reflect the robustness of the analysis.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5951454/v1/b3702ac4cf7f9032ec3788b0.jpg"},{"id":84726862,"identity":"0da2e8f9-4e0f-49d7-b50b-df4f9594e89e","added_by":"auto","created_at":"2025-06-16 16:08:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1259908,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5951454/v1/3eda7a0c-7ba9-4b13-b5d0-1fd14aa159af.pdf"},{"id":81410545,"identity":"68d48ad8-aa49-4a5d-9fe5-7a8dde8389bf","added_by":"auto","created_at":"2025-04-25 20:21:58","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20955,"visible":true,"origin":"","legend":"","description":"","filename":"NdioneetalXenosuveillanceSupplementaryTable1OneHealthOutlookID031e2a71e5364a5d9f2ddad3d1b997bd.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5951454/v1/91c52b926b3ee87bffffc412.xlsx"},{"id":81410546,"identity":"f034022a-7bb6-4314-9148-3d759fe2bec6","added_by":"auto","created_at":"2025-04-25 20:21:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13826,"visible":true,"origin":"","legend":"","description":"","filename":"NdioneetalXenosuveillanceSupplementaryTable2OneHealthOutlookID031e2a71e5364a5d9f2ddad3d1b997bd.docx","url":"https://assets-eu.researchsquare.com/files/rs-5951454/v1/ab9c375aaef12f9bc92bd328.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mosquito-Based Detection of Endogenous Jaagsiekte Sheep Retrovirus in Senegal: Expanding the Scope of Xenosurveillance","fulltext":[{"header":"Background","content":"\u003cp\u003eThe discovery of viruses transmitted by arthropod vectors has transformed our understanding of viral ecology and infectious disease dynamics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mosquitoes, in particular, play a critical role in transmitting a wide range of pathogens that affect both animal and human populations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This fact has prompted extensive research into the complex interactions between vectors, viruses, and hosts, with a special focus on zoonotic spillover events where pathogens cross species barriers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Advances in next-generation sequencing (NGS) technologies have further accelerated pathogen discovery, especially in the context of vector-borne diseases. NGS enables high-throughput, unbiased sequencing, allowing for the detection of both known and novel viruses in diverse ecological settings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXenosurveillance, or the sampling of pathogen genetic material from the blood meals of arthropod vectors, has proven effective for detecting viruses circulating in wildlife and domestic animals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. When combined with metagenomic sequencing, xenosurveillance offers comprehensive insights into viral diversity in specific environments. This approach not only aids in the discovery of new pathogens but also helps clarify viral transmission ecology, identifying potential vectors and reservoirs for zoonotic diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eJaagsiekte sheep retrovirus (JSRV) is a betaretrovirus known to cause ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer in sheep [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Transmission occurs primarily via aerosolized droplets, and the virus has been considered tightly restricted to sheep and closely related ruminants. Two forms of JSRV exist: the exogenous, infectious virus (exJSRV), and multiple endogenous variants (enJSRVs), which are stably integrated into the host genome. These endogenous forms share high sequence similarity with exJSRV and retain some functional elements, raising interest in their ecological relevance and evolutionary relationship. Traditionally, arthropods have not been implicated in JSRV transmission, and no vector-borne involvement has been demonstrated.\u003c/p\u003e \u003cp\u003eIn this study, alongside other arthropod-borne and non-arthropod-borne viruses, we report the first detection of JSRV-related genetic material in blood-fed mosquitoes collected in southeastern Senegal, identified through a vector-enabled metagenomic approach. Phylogenetic analysis revealed that the sequence is most closely related to enJSRV. While this finding does not indicate active viral replication or vector-borne transmission, it provides indirect evidence of mosquito contact with local ruminant hosts, particularly sheep. This supports the growing utility of xenosurveillance for capturing host-derived genetic signatures, in addition to circulating pathogens. Moreover, it aligns with emerging ecological observations, such as the recent detection of JSRV RNA in surface water from Tanzanian waterholes during dry seasons [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which together raise important questions about non-traditional interfaces of viral exposure and environmental persistence in pastoral ecosystems.\u003c/p\u003e \u003cp\u003eBy capturing host-derived retroviral sequences through mosquito metagenomics, our work underscores the broader potential of xenosurveillance to advance understanding of host\u0026ndash;vector\u0026ndash;environment interactions and to support integrated pathogen and exposure monitoring systems at the intersection of animal and public health.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites\u003c/h2\u003e \u003cp\u003eBlood-fed mosquitoes were collected between 2016 and 2019 from three ecologically significant regions in Senegal, Louga in the northwest, Barkedji in the central Ferlo region, and Kedougou in the southeast, each chosen for their relevance to vector-borne disease dynamics. Louga serves as a transitional zone between agriculture and pastoralism, where fluctuating rainfall and rising temperatures impact livestock and crop production, fostering mosquito-host interactions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Barkedji, centrally located, features pastoral corridors and wildlife reserves, providing a critical interface for zoonotic spillovers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Pastoral activities in the region are marked by seasonal livestock movements, with sheep and goats forming the backbone of herds due to their resilience to water scarcity. Kedougou lies within a tropical forest zone near the Guinean border, where the humid climate supports high mosquito activity, making it a hotspot for pathogen circulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although pastoralism is less dominant in this area compared to the Ferlo or Louga regions, Kedougou has become an important destination for herders from northern Senegal due to its abundant pasture and water resources, especially during dry seasons. Herders are drawn to Kedougou for economic opportunities, including livestock markets where animals command attractive prices, enabling them to diversify their income sources [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMosquito collection and sample selection\u003c/h3\u003e\n\u003cp\u003eThis study analyzed archived mosquito pools collected biweekly between 2016 and 2019 as part of the national arbovirus surveillance program coordinated by IPD. Mosquitoes were collected using a variety of trapping methods described by Ndiaye et al., including CDC light traps baited with CO₂, placed at both ground and canopy levels [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Traps were deployed near livestock enclosures, village edges, and natural water sources in ecologically diverse regions such as Barkedji, Louga, and Kedougou.\u003c/p\u003e \u003cp\u003eFollowing morphological identification on the field, mosquito pools were archived at the Centre de Recherche sur les Arbovirus (CRORA) biobank [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For this study, a subset of pools was pre-selected based on their ability to induce cytopathic effect (CPE) upon inoculation in mosquito and mammal cell culture but had tested negative by indirect immunofluorescence assay (IFA) for common arboviruses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This selection strategy prioritized samples likely to contain uncharacterized or non-target viral agents.\u003c/p\u003e \u003cp\u003eWhole mosquito specimens from the selected pools were homogenized, and total nucleic acids were extracted for metagenomic sequencing to identify both host- and pathogen-derived viral material.\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and Metagenomic Sequencing\u003c/h3\u003e\n\u003cp\u003eRNA extraction was performed using the MagMax Viral/Pathogen Nucleic Acid Isolation Kit and the KingFisher Flex Purification System (Thermo Fisher), following the manufacturer\u0026rsquo;s recommendations. Sequencing was conducted on the extracted RNA using an Illumina-based unbiased approach, as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, host ribosomal RNA was depleted using specific rRNA-targeting probes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and RNase H, and the remaining RNA served as the template for first-strand cDNA synthesis with the SuperScript IV Reverse Transcriptase kit. Double-stranded cDNA was then generated using Klenow fragment DNA polymerase. Libraries were prepared with the Nextera XT DNA Library Preparation kit (Illumina), and sequencing was carried out on the NextSeq 550 system.\u003c/p\u003e \u003cp\u003eThe resulting sequences were analyzed using the CZ-ID metagenomics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://czid.org\u003c/span\u003e\u003cspan address=\"http://czid.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 15 March 2023) for viral identification [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Default pipeline parameters were used, incorporating quality control steps such as adapter trimming, low-complexity read filtering, and the removal of host-derived reads (sheep, goat, human) as well as common laboratory contaminants. Sequence alignment was performed against curated nucleotide (NT) and protein (NR) databases using Bowtie2 and RAPSearch2, respectively. Reads were assembled into contigs with SPAdes, and taxonomic classification was based on nucleotide and translated alignments, applying identity thresholds of approximately 80\u0026ndash;85% for NT and 90\u0026ndash;95% for NR, with e-value cutoffs set at \u0026le;\u0026thinsp;1e-10.\u003c/p\u003e \u003cp\u003eAdditionally, the assembled sequences were subjected to BLAST analysis (Basic Local Alignment Search Tool) against the NCBI nucleotide database to confirm viral identity and assess sequence similarity with known viral genomes, facilitating more precise taxonomic classification and strain identification.\u003c/p\u003e\n\u003ch3\u003eMolecular Assay Development\u003c/h3\u003e\n\u003cp\u003eTo confirm the presence of JSRV detected through NGS, we developed two TaqMan-based reverse transcription quantitative PCR (RT-qPCR) assay were developed. These assays utilized primers and a fluorescently labeled probe designed with Primer3 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], specifically targeting regions of the envelope gene and the 3' untranslated region (3\u0026rsquo;UTR) based on our assembled JSRV sequences. The designed oligonucleotide sets were rigorously evaluated in silico for specificity using BLAST analysis, ensuring that they would exclusively bind to the target JSRV sequences without cross-reactivity with other viral or host genomes. In addition to the in-house RT-qPCR, we employed the hemi-nested U3hn PCR, a previously described method targeting the U3 region of the viral long terminal region (LTR), and known for its high sensitivity in field studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This assay was used to complement and validate the results obtained with our newly developed RT-qPCR assay. All assays were performed on RNA extracted from mosquito pools, providing a robust confirmation framework for the detection of JSRV-related sequences.\u003c/p\u003e\n\u003ch3\u003eJaagsiekte virus Phylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eThe consensus data generated during this study were aligned with a representative dataset of available sequences using the multiple sequence alignment tool MAFFT (version 7) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] with default parameters. This alignment allowed for a comprehensive comparison of the newly detected sequence with known viral genomes, providing a robust framework to establish their evolutionary relationships. The aligned sequences were then used to construct a maximum likelihood (ML) phylogenetic tree, employing IQ-TREE [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], a widely recognized tool for phylogenetic inference. The best-fit substitution model for the analysis was selected using ModelFinder [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], ensuring an accurate representation of the evolutionary processes underlying the data. The phylogenetic tree was visualized using FigTree (version 1.4.4) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tree.bio.ed.ac.uk/software/figtree/\u003c/span\u003e\u003cspan address=\"http://tree.bio.ed.ac.uk/software/figtree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 17 March 2023), a flexible software for exploring and presenting phylogenetic results.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eViral Diversity in Mosquito Populations, Co-detection Patterns and Regional Variation\u003c/h2\u003e \u003cp\u003eA total of 78 mosquito pools collected between 2016 and 2019 were included in the metagenomic analysis. Although our primary focus was on blood-fed female mosquitoes, some pools also included non-blood-fed females and a few males due to the retrospective nature of the archived material. These pools were derived from diverse mosquito species and sites across Senegal.\u003c/p\u003e \u003cp\u003eOur metagenomic sequencing identified a wide array of viruses across various mosquito species collected from different regions of Senegal between 2016 and 2019 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These included both arboviruses and non-arboviruses, demonstrating the remarkable diversity of viral communities harbored by mosquitoes. Notable detections were observed in species such as \u003cem\u003eAedes aegypti\u003c/em\u003e, \u003cem\u003eAedes vexans\u003c/em\u003e, \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, and \u003cem\u003eAedes furcifer\u003c/em\u003e, each playing a distinct role in the viral ecosystem. The identified viruses spanned multiple viral species, with significant detections of Pestivirus A, Dezidougou virus, and Culex iflavivirus, reflecting a broad spectrum of viral types circulating in the environment. Co-detections were frequently observed across several mosquito species, suggesting complex viral interactions within vector populations. While multiple viruses were identified in some pooled samples, we refer to these as instances of co-detection rather than confirmed co-infections. Given that each pool may contain multiple mosquitoes, the detected viral sequences could originate from different individuals. As such, these findings reflect aggregate viral presence within the pool and do not indicate concurrent infection of a single mosquito.\u003c/p\u003e \u003cp\u003eThe metrics associated with viral identification are provided in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMosquito Species and Viruses Identified Across Collection Sites (2016\u0026ndash;2019)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026minus;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMosquito species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCollection locality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDate of Collection (Year-Month)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIdentified Viruses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNumber of pools\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNumber of individuals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFeeding Status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLouga\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2017-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAedes Albopictus Densovirus / Dezidougou virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2017-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eKedougou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A / Taesano Aedes Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTaesano Aedes Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAedes centropunctatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKedougou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eAedes dalzieli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKedougou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAedes furcifer\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKedougou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A /Mosquito Densovirus BR/07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAedes ochraceus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eAedes sudanensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou Virus / Daezongdong Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAedes taylori\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKedougou\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eAedes unilineatus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eBarkedji\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e2019-09\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eJaagsiekte Sheep Retrovirus\u003c/b\u003e / Dezidougou Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eFed-blood\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cem\u003eAedes vexans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2016-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou virus / Aedes vexans iflavivirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2019-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e2019-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAedes Vexans iflavivirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAedes Vexans iflavivirus / Dezidougou Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eAnopheles pharaonsis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cem\u003eCulex quinquefasciatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eLouga\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2017-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCulex iflavivirus / Pestivirus H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHubei partiti-like Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHubei partiti-like Virus / Avian Leukosis Virus / Yongsan iflavivirus/Culex iflavivirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2017-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCulex iflavivirus / Avian leukosis Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e2017-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAedes Albopictus Densovirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCulex flavivirus /Culex Densovirus/ Mosquito Densovirus BR/07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus H / Hubei mosquito virus / Culex iflavivirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCulex tritaeniorhyncus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLouga\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2017-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCulex antennatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eUnfed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eCulex ethiopicus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2016-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2019-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A / Dezidougou Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCulex neavei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBarkedji Virus / Bagaza Virus / Sindbis Virus / West-Nile Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBarkedji Virus / Bagaza Virus / Usutu Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCulex perfuscus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2016-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cem\u003eCulex poicilipes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e2019-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou Virus / Aedes vexans iflavivirus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou Virus / Daezongdong Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eCulex quinquefasciatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2019-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAedes Pseudocutellaris Reovirus / Pestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDezidougou Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePestivirus A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFed-blood\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCulex tigripes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2017-08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhlebotom sp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBarkedji\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c4\"\u003e \u003cp\u003e2019-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor example, Pestivirus A, known as bovine viral diarrhea virus type 1 infecting various vertebrates including cattle, sheep, goats, pigs and wild ruminants was widely detected in mosquitoes from multiple regions, including Kedougou and Barkedji, particularly in species like \u003cem\u003eAedes unilineatus\u003c/em\u003e and \u003cem\u003eAedes furcifer\u003c/em\u003e. Dezidougou virus, another frequently identified virus, was found alongside Aedes vexans iflavivirus in \u003cem\u003eAedes vexans\u003c/em\u003e pools from Barkedji. In \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e from Louga, co-detections were observed, with Culex iflavivirus and Pestivirus H appearing in the same pools as Hubei partiti-like virus and Avian Leukosis virus, highlighting the complexity of viral ecosystems in the region.\u003c/p\u003e \u003cp\u003eThese findings underscore the extensive viral diversity present within mosquito populations, with marked regional variation observed across the study sites. Distinct viral profiles were found in Barkedji (51 pools, 1,085 mosquitoes), Louga (19 pools, 192 mosquitoes), and Kedougou (8 pools, 70 mosquitoes), likely influenced by local ecological factors. Barkedji and Louga each harbored 13 unique viral species, while Kedougou, a tropical forest site, had only 3. This may reflect our sampling strategy, which focused on pools that induced cytopathic effects in mosquito or mammalian cell cultures but tested negative for common arboviruses, enhancing the detection of uncharacterized or non-target viruses. Such variation points to the critical role of regional climate, host availability, and habitat conditions in shaping mosquito-associated viral communities.\u003c/p\u003e \u003cp\u003eNotably, co-detections within the same mosquito pools were more common in samples from Louga and Barkedji, suggesting that these regions may be hotspots for viral transmission and co-circulation. While we cannot infer true co-infections at the individual mosquito level due to the use of pooled samples, these patterns may reflect overlapping ecological niches or shared host exposure, facilitating the simultaneous presence of multiple viral species in the same sampling units.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetection of Jaagsiekte Sheep Retrovirus\u003c/h3\u003e\n\u003cp\u003eAmong the various viruses identified, our analysis revealed the first detection of JSRV in mosquitoes. From a blood-fed mosquito pool collected in Barkedji in 2019, we recovered a partial genome sequence (about 600 base pairs in length) encompassing the terminal region of the \u003cem\u003eEnv\u003c/em\u003e gene and 3'UTR of JSRV. Based on these findings, we developed two sets of molecular assays to confirm the taxonomic identity of JSRV (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eJaagsiekte sheep retrovirus-targeted RT-qPCR assays designed during the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRT-PCR set 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eEnveloppe gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJaagsiekteV_Left primer_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCGGTTCTGACTGTTGTGCTT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJaagsiekteV_Right primer_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCGCAGCTCCCCTCTCTTTAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJaagsiekteV_Probe_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFAM - AACATGTTGCAACACCGACA - BBQ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRT-PCR set 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3' untranslated region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJaagsiekteV_Left primer_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCCTAAGCTCCCTGTCCCG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJaagsiekteV_Right primer_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGCCTTCCTTTATTGTGCTGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJaagsiekteV_Probe_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFAM - TGTGAATGTCAGAAGTCACGT - BBQ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further support these results, we also employed the previously validated U3hn PCR to complement our in-house assays. The U3hn PCR served as an independent molecular confirmation of JSRV presence, particularly useful in low-viral-load samples.\u003c/p\u003e \u003cp\u003eRT-qPCR and U3hn PCR were both performed on the mosquito pool in which JSRV was initially detected, along with all nine additionally blood-fed pools with no prior evidence of infection. The assays confirmed JSRV presence in the original mosquito pool (Supplementary table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), though follow-up sequencing did not yield additional coverage of the viral genome.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic Analysis of Jaagsiekte Sheep Retrovirus\u003c/h2\u003e \u003cp\u003eTo further characterize the JSRV-related sequence detected in mosquitoes, we performed a phylogenetic analysis using a multiple sequence alignment covering a region of the partial Env gene and the LTR. This alignment included reference betaretroviruses, with particular emphasis on both exJSRV and endogenous (enJSRV) JSRV sequences. A ML phylogenetic tree was then constructed to determine the evolutionary placement of the mosquito-derived sequence.\u003c/p\u003e \u003cp\u003eThe resulting tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) revealed that the sequence clusters closely with enJSRV strains integrated in the sheep genome, rather than with known exogenous, infectious variants (exJSRV). High bootstrap support (\u0026gt;\u0026thinsp;90%) on key branches further confirms the robustness of these evolutionary relationships.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides significant insights into the viral ecosystems within mosquito populations in Senegal, identifying a diverse array of viruses, including Pestivirus A, Dezidougou virus, and Culex iflavivirus. The extensive detection of these viruses across various mosquito species, underscores the complexity of viral interactions within vector populations. In the context of xenosurveillance, platforms like CZ-ID provide a standardized approach for detecting viral sequences in blood-fed mosquitoes. However, reliance on default parameters may reduce sensitivity to highly divergent or novel viruses, particularly in ecologically rich settings, potentially leading to an underestimation of viral diversity, a limitation we acknowledge in our interpretation. These findings highlight the critical role that mosquitoes play, not only as vectors of well-established arboviruses but also as reservoirs or passive carriers of non-arboviral pathogens [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This emphasizes the need for broader surveillance efforts to understand the contributions of mosquito populations to viral ecology and pathogen transmission.\u003c/p\u003e \u003cp\u003eThe detection of JSRV-related genetic material in mosquitoes through unbiased metagenomic sequencing adds a novel dimension to vector-based surveillance. Phylogenetic analysis indicates that the detected sequence is most closely related to enJSRV, non-infectious retroviral elements integrated into the sheep genome. While this does not suggest active viral replication or vector-borne transmission, it provides indirect molecular evidence of mosquito feeding on local ruminant hosts, particularly plausible in Barkedji, a pastoral region characterized by seasonal livestock movements and a high density of sheep and goats. This finding highlights the potential of mosquitoes to act as biological samplers, capturing host-derived viral sequences through bloodmeals, and supports the application of xenosurveillance for ecological monitoring of livestock exposure.\u003c/p\u003e \u003cp\u003eThe detection of JSRV-related sequences in mosquitoes also aligns with prior findings from Nigeria and Cameroon, where JSRV-related sequences were identified in human samples [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While the biological relevance of those findings remains uncertain, they collectively raise important questions about the distribution and detectability of endogenous retroviral elements across species and environments [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In our study, the identification of enJSRV-like sequences in mosquito bloodmeals, alongside the detection of other viruses such as Pestivirus A, Dezidougou virus, and Culex iflavivirus, highlights the value of combining xenosurveillance and metagenomic tools to monitor both actively circulating viruses and genomic material derived from host organisms. This approach provides a window into local livestock exposure and virus\u0026ndash;host\u0026ndash;vector interactions without the need for direct sampling of animals. Further investigation is warranted to better understand the ecological roles of retroviruses like enJSRV and their incidental detection in non-host organisms such as arthropod vectors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Previous studies in Senegal have reported the presence of ovine betaretroviruses, such as ENTV, directly in infected tissues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. By expanding this lens to vector-based environmental surveillance, our findings underscore the potential of mosquitoes to act as passive carriers and ecological sentinels, capturing signals of both active infections and host genomic signatures that may otherwise go undetected.\u003c/p\u003e \u003cp\u003eThe utility of xenosurveillance as a tool for pathogen detection is further exemplified in this study. By analyzing viral genetic material obtained from blood-feeding mosquitoes, we were able to identify viruses circulating within local wildlife and domestic animal populations without the need for direct mammalian sampling. This method offers a less invasive and more practical approach to surveillance, particularly in remote or logistically challenging regions. Xenosurveillance not only expands our understanding of viral geographic distribution and host interactions but also enhances the early detection of emerging zoonotic threats [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This approach is especially relevant in areas with high biodiversity and frequent interactions between wildlife, domestic animals, and humans, such as Senegal.\u003c/p\u003e \u003cp\u003eBeyond its contributions to pathogen discovery, this study emphasizes the critical role of molecular diagnostics in enhancing viral surveillance. The development of specific molecular assays targeting JSRV-related sequences, informed by NGS data, enabled us to confirm the presence of endogenous retroviral material in mosquito samples. This integration of high-throughput sequencing with targeted molecular tools exemplifies a robust approach not only for pathogen detection but also for identifying host-derived genetic signatures within vector populations. It also underscores the importance of adapting diagnostic approaches to capture both arboviral and non-arboviral elements, thereby expanding the scope of mosquito-based surveillance beyond classical vector-borne disease paradigms [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The implications of this study extend to public health preparedness, as the early detection of emerging viral threats can inform targeted interventions and outbreak prevention strategies.\u003c/p\u003e \u003cp\u003eUltimately, this work highlights the transformative potential of integrating xenosurveillance with next-generation sequencing and molecular diagnostics to enhance our understanding of virus-vector-host interactions. By detecting host-derived endogenous retroviral sequences in mosquito bloodmeals, xenosurveillance can provide insight into both pathogen presence and host exposure, capturing ecological contact patterns that extend beyond classical infection-based surveillance. This approach offers a scalable and minimally invasive tool for monitoring livestock health and supporting early-warning systems at the intersection of animal and public health, particularly in pastoral settings where direct sampling remains a challenge. As global health systems move toward integrated One Health frameworks, mosquito-based xenosurveillance offers a unique opportunity to strengthen disease surveillance across species and environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed in this study are provided within the article and its supplementary materials, while the genomic data are publicly accessible in the NCBI repository.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the NIH West African Center for Emerging Infectious Diseases [grant number U01AI151801-01], the NIH PICREID [grant number U01AI151758] and the Africa CDC Pathogen Genomics Initiative funds [CARES grant 4306-22-EIPHLSSGENOMICS].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMHDN, MD, SS, MK, MM, SMBSD, SFMD and MMD contributed to the laboratory experiments; EHN, BD, DD, NMS, FAS and AG contributed to the field sampling and species identification; MHDN, AG and MMD designed the study; MHDN, EHN, AG and MMD contributed to the data analysis and curation; AAS, OF, ND, SCW, OumF, MawD, GF and MMD provided administrative and logistical support; MHDN, EHN, AG and MMD drafted the manuscript; all authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the technical staff of the Institut Pasteur de Dakar for their invaluable support in during this work. 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Front Microbiol. 2019;10:22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2019.00022\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.00022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"one-health-outlook","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oneh","sideBox":"Learn more about [One Health Outlook](https://onehealthoutlook.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/oneh/default.aspx","title":"One Health Outlook","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Jaagsiekte sheep Retrovirus, xenosurveillance, mosquitoes, Senegal, metagenomics, virus–host–vector interactions","lastPublishedDoi":"10.21203/rs.3.rs-5951454/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5951454/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMosquitoes are well-known vectors for arthropod-borne viruses, yet their role as passive carriers of non-arthropod-borne viruses remains underexplored. Xenosurveillance, a method that utilizes blood-feeding arthropods to sample host and pathogen genetic material, has emerged as a valuable tool in viral ecology. In this study, we report the first identification of Jaagsiekte Sheep Retrovirus (JSRV)-related sequences in blood-fed mosquitoes collected in Senegal. JSRV, a betaretrovirus responsible for ovine pulmonary adenocarcinoma, is typically found in sheep, but its genetic trace in mosquitoes offers a novel perspective on host\u0026ndash;vector contact and surveillance. Our study aimed to investigate whether mosquitoes can serve as sentinels for detecting both pathogens and host-derived markers in complex ecosystems.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMosquitoes were collected between 2016 and 2019 from three ecologically significant regions in Senegal (Louga, Barkedji, and Kedougou). Blood-fed mosquitoes were pooled and subjected to RNA extraction and metagenomic sequencing using Illumina NextSeq550. Sequencing data were analyzed with CZ-ID and BLAST for viral identification. RT-qPCR assays were designed to validate the presence of JSRV-related sequences, targeting conserved regions of the envelope gene and 3' untranslated region. Phylogenetic analysis was conducted using MAFFT and IQ-TREE to compare the detected sequence with global exogenous and endogenous JSRV references.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA diverse array of viruses across mosquito species, including both arboviruses and non-arthropod-borne viruses. A JSRV-related sequence was detected in a single blood-fed mosquito pool collected in Barkedji (2019). The RT-qPCR assay confirmed JSRV presence, validating the sequencing results. Phylogenetic analysis revealed strong similarity to known endogenous JSRV (enJSRV) sequences integrated in the sheep genome, indicating that the detected material likely originated from host DNA ingested during blood feeding.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eThis study presents the first report of endogenous retroviral sequences detected in mosquitoes, alongside the identification of actively circulating viruses, highlighting the broader potential of mosquitoes as environmental sentinels. While mosquitoes are not biological vectors for JSRV, their ability to capture both host-derived retroviral material and pathogenic viral genomes through bloodmeals reinforces the value of xenosurveillance for monitoring livestock\u0026ndash;vector\u0026ndash;environment interactions. These findings contribute to broader efforts in integrated disease surveillance and underscore the utility of combining metagenomics with molecular diagnostics to detect diverse viral signals in high-risk ecological settings.\u003c/p\u003e","manuscriptTitle":"Mosquito-Based Detection of Endogenous Jaagsiekte Sheep Retrovirus in Senegal: Expanding the Scope of Xenosurveillance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 20:21:53","doi":"10.21203/rs.3.rs-5951454/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-23T12:01:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T10:19:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-01T21:08:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81976133945517705190732129117495977470","date":"2025-05-01T21:03:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-28T09:55:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171585898577570245512422768477243652395","date":"2025-04-27T21:19:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228688003899795127017761798026998718557","date":"2025-04-24T11:02:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-24T10:02:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-16T13:45:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"One Health Outlook","date":"2025-04-12T21:47:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"one-health-outlook","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oneh","sideBox":"Learn more about [One Health Outlook](https://onehealthoutlook.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/oneh/default.aspx","title":"One Health Outlook","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c82cb78e-f667-4b88-a5f4-362fba0013bb","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:06:45+00:00","versionOfRecord":{"articleIdentity":"rs-5951454","link":"https://doi.org/10.1186/s42522-025-00155-7","journal":{"identity":"one-health-outlook","isVorOnly":false,"title":"One Health Outlook"},"publishedOn":"2025-06-10 15:57:42","publishedOnDateReadable":"June 10th, 2025"},"versionCreatedAt":"2025-04-25 20:21:53","video":"","vorDoi":"10.1186/s42522-025-00155-7","vorDoiUrl":"https://doi.org/10.1186/s42522-025-00155-7","workflowStages":[]},"version":"v1","identity":"rs-5951454","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5951454","identity":"rs-5951454","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}