Metagenomic Characterization of Aedes aegypti Virome in Kwale County, Kenya

Aedes aegypti, Metagenomics, Insect-specific viruses 13 14 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint

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Aedes aegypti is a primary vector of arboviruses, including dengue, 16 chikungunya, yellow fever, and Zika, and is widespread along the Kenyan coast, a region with 17 recurrent outbreaks. In addition to human -pathogenic arboviruses, Ae. aegypti harbors insect-18 specific viruses (ISVs) that replicate exclusively in arthropods and may influence mosquito 19 physiology, immunity, and vector competence. However, data on ISVs in Kenyan Ae. aegypti 20 populations are limited. 21

Twenty-nine mosquito pools containing 20 individual mosquitoes from Kwale 22 County were homogenized, combined into a superpool, and subjected to total RNA extraction. 23 Libraries were sequenced on an Illumina MiSeq platform. Initial analysis was done on CZ -ID 24 platform. Reads were quality -filtered using PrinseqLite and assembled de novo with 25 MEGAHIT. Phylogenetic analyses was performed with IQ -TREE using the Maximum 26 Likelihood method. 27

Metagenomic analysis revealed diverse ISVs in Ae. aegypti. Complete genomes of 28 Fako virus and Tesano Aedes virus, and partial genomes of Aedes partiti-like virus, Cell fusing 29 agent virus, and Formosus virus, were recovered. Genome lengths ranged from 1,149 to 10,146 30 nucleotides, with 91 –99.5% identity to reference strains. Phylogenetic analysis placed the 31 viruses within established ISV lineages, showing close evolutionary relationships with strains 32 previously reported from Africa and other regions. 33

This study provides comprehensive characterization of ISVs in Ae. aegypti from 34 Kwale County. The recovery of complete and near -complete genomes demonstrates the 35 diversity and active circulation of ISVs, establishing a baseline for future studies on mosquito 36 viromes, virus–mosquito interactions, and potential impacts on vector competence. 37 . 38 39 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Importance 40 Insect-specific viruses (ISVs) are widespread in mosquitoes and replicate exclusively in 41 arthropods, influencing mosquito physiology, immunity, and potentially vector competence for 42 human-pathogenic arboviruses. Despite recurrent arboviral outbreaks along the Kenyan coast, 43 the diversity and ecological roles of ISVs in Aedes aegypti remain poorly understood . This 44 study provides a comprehensive metagenomic characterization of ISVs in Ae. aegypti from 45 Kwale County, revealing complete genomes of Fako virus and Tesano Aedes virus, along with 46 near-complete genomes of Aedes partiti-like virus, Cell fusing agent virus, and Formosus virus. 47 These findings establish a baseline for mosquito virome composition in this region and 48 underscore the active circulation of diverse ISVs in natural populations. Understanding these 49 virus–mosquito interactions is critical for interpreting arbovirus ecology, assessing the 50 potential impacts of ISVs on vector competence, and informing future vector surveillance and 51 biological control strategies. 52 53 54 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint

55 Aedes aegypti (Diptera: Culicidae) is a primary mosquito vector responsible for the 56 transmission of several medically important arboviruses, including dengue virus (DENV), Zika 57 virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV) (Omuoyo et al., 58 2023). These vector -borne pathogens represent significant public health challenges globally 59 and in Kenya, where repeated outbreaks of dengue fever and other Aedes-transmitted diseases 60 have been documented along the coastal and wester n regions (Courtney & Cranston, 2015) . 61 The ecology of Ae. aegypti is shaped by its adaptability to urban and peridomestic 62 environments, high anthropophily, and ability to exploit diverse breeding habitats, making it a 63 persistent and efficient arbovirus vector (Bhatt et al., 2013). 64 In addition to their role in transmitting vertebrate-infecting arboviruses, Ae. aegypti mosquitoes 65 harbor a diverse assemblage of insect-specific viruses (ISVs), which are viruses that replicate 66 exclusively in insects and are unable to infect vertebrate hosts (Carvalho & Long, 2021; Oguzie 67 et al., 2022; Patterson et al., 2020) . ISVs have been identified in multiple viral families, 68 including Flaviviridae, Togaviridae, and others, reflecting extensive diversity within mosquito 69 viromes (Amoa-Bosompem et al., 2020). The first ISV ever described was the cell fusing agent 70 virus (CFAV), isolated from an Ae. aegypti cell culture, which does not replicate in vertebrate 71 cells and is widely regarded as the prototype insect-specific virus (Bolling et al., 2015; Martin 72 et al., 2019) . Since then, advances in high -throughput sequencing and metagenomics have 73 greatly expanded the catalogue of ISVs associated with mosquitoes (Langat, 2023) . 74 Emerging evidence suggests that ISVs may influence mosquito biology and vector competence 75 in complex ways. Some ISVs have been shown to modulate arbovirus replication, potentially 76 through mechanisms such as superinfection exclusion or interactions with the mosquito 77 immune system (Nasar et al., 2012; Vasilakis et al., 2013; Vasilakis & Tesh, 2015) . Although 78 the precise effects of many ISVs on arbovirus transmission are not fully resolved, experimental 79 studies indicate that ISVs can either suppress or alter the replication dynamics of co -infecting 80 arboviruses in mosquito hosts, with implications for d isease transmission (Fish et al., 2017; 81 Parry et al., 2021). These interactions highlight the importance of considering the broader viral 82 ecology within mosquito populations when evaluating vector competence and arbovirus 83 transmission risk. 84 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Insect-specific viruses have increasingly attracted attention for their potential use in the 85 development of novel vaccines. Because ISVs replicate exclusively in insect cells and are non-86 pathogenic to vertebrates, they provide a safe platform for the production of viral antigens and 87 virus-like particles (VLPs) for immunization purposes (Erasmus et al., 2018; Hall-Mendelin et 88 al., 2016; Hall et al., 2025) . Recombinant ISVs expressing arboviral structural proteins have 89 been shown to elicit protective immune responses in animal models without the risk of causing 90 disease in humans (Nasar et al., 2015; Tan et al., 2023) . Such approaches could serve as 91 scalable and safe alternatives to conventional live -attenuated or inactivated vaccines, 92 particularly for arboviruses such as dengue, Zika, and chikungunya that remain significant 93 public health threats in endemic regions including Kenya. 94 95 In addition to their ecological roles, ISVs have significant potential in the development of 96 ELISA-based diagnostic tools for arbovirus surveillance (Erasmus et al., 2015) . Because 97 ISV-based chimeric constructs can express structural antigens of vertebrate -infecting viruses 98 while remaining replication-restricted in vertebrate cells, they provide a safe alternative antigen 99 source for use in immunoassays. For example, chimeras based on the insect -restricted Eilat 100 virus have been successfully used as high -quality antigen in IgM ELISA formats for 101 chikungunya virus, demonstrating sensitivity and specificity comparable to traditional assays 102 while allowing handling at lower biosafety levels and reducing reliance on live pathogenic 103 virus preparations (Erasmus et al., 2015). This approach enhances the feasibility of developing 104 ELISA diagnostics that can be deployed in resource-limited settings, improve assay safety, and 105 reduce costs associated with antigen production. ISV -based antigens may thus strengthen 106 serological surveillance frameworks for arboviruses by providing robust, safe, and scalable 107 tools for early detection and outbreak monitoring 108 Despite growing interest in the ecological and evolutionary roles of ISVs, genomic data and 109 detailed characterization of these viruses from Ae. aegypti populations in East Africa remain 110 limited. Metagenomic studies in Kenya have identified a variety of ISVs, including 111 flavivirus-like agents and iflaviruses, underscoring the presence of diverse viral taxa 112 co-circulating with pathogenic arboviruses in local mosquito populations (Chiuya et al., 2021; 113 Langat et al., 2021; Omuoyo et al., 2023) . However, comprehensive genomic analyses and 114 phylogenetic studies of Kenyan ISVs are still scarce, leaving gaps in our understanding of their 115 diversity, evolutionary relationships, and potential functional interactions with arboviruses. By 116 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint documenting the genomic diversity and evolutionary relationships of ISVs, studies such as ours 117 not only enrich baseline virome resources but also offer avenues for exploiting ISVs in public 118 health interventions. These findings have implications for arboviral risk assessment, vector 119 surveillance programs, and the design of innovative vector control and disease mitigation 120 strategies. In regions like coastal Kenya, where Ae. aegypti -driven arboviral outbreaks are 121 recurrent, understanding the interplay between ISVs and pathogenic arboviruses could 122 ultimately contribute to reducing disease burden and improving outbreak preparedness. 123 124 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Methodology 125 Ethical Approval 126 Ethical approval was obtained from the Kenya Medical Research Institute (KEMRI) Scientific 127 and Ethics Review Unit (SERU) under protocol number KEMRI/SERU/C VR/4702 and 128 WRAIR# 3101. Permission to conduct the study was granted by the National Council for 129 Science, Technology, and Innovation (NACOSTI). 130 Study area 131 The study was conducted in Kwale County, (4.1730°S, 39.4520°E) which lies along the coastal 132 region with a tropical climate, high temperatures (26 –32°C), and seasonal rainfall that create 133 favorable conditions for Aedes aegypti proliferation. 134 135 Figure 1. Map of the Kenyan coast showing mosquito sampling sites in Kwale County. Base 136 maps, boundaries and shape files of Kenyan map and administrative boundaries of the Counties 137 were derived from GADM data version 4.1 ( https://gadm.org) and the maps were generated 138 using ArcGIS Version 10.2.2 ( http://desktop.arcgis.com/en/arcmap) advanced license) 139 courtesy of Samuel Owaka. 140 Entomological Investigation 141 Sampling of adult mos quitoes was conducted between 12 th March and 21 st June 2022 . 142 Mosquitoes were collected using CDC miniature light traps (Model 512, John Hock Co., 143 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Gainesville, Florida, USA). Traps baited with carbonated dry ice (CO2) were deployed 144 overnight (6pm-6am) in favourable habitat, including dwelling quarters and animal sheds. The 145 samples were linked to the sites by geo-coding using a GPS. The mosquitoes were immobilized 146 by freezing at -20ºC for 20mins and identified morphologically to species under a dissecting 147 microscope using taxonomy keys, including Edwards (1941) (Road & Quaritch, 1936) , 148 Harbach (1988)(Harbach, 1988) and Jupp (1986) (Jupp, 1986). The identified mosquitoes were 149 pooled in groups of 1 to 20 samples based on species, sex and collection site. Mosquitoes were 150 consequently preserved in liquid nitrogen, and transported to the laboratory at the Kenya 151 Medical Research Institute in Kisumu, where they were stored at-80˚C until further processing. 152 153 Mosquito sample preparation 154 A total of 580 mosquitoes collected from Kwale County were pooled into 29 pools, with each 155 pool comprising 20 individual mosquitoes . The pools were homogenized using a Mini-156 Beadruptor-16 (BioSpec Products, Bartlesville, OK, USA) in 1,000 µL of homogenization 157 medium, consisting of minimum essential medium supplemented with 15% fetal bovine serum 158 (FBS) (Gibco, Life Technologies, Grand Island, NY, USA), 2% L-glutamine (Sigma-Aldrich), 159 and 2% antibiotic–antimycotic solution (Gibco, Life Technologies), together with zirconium 160 beads (2.0 mm diameter) for 40s. Homogenates were subsequently centrifuged at 10,000 rpm 161 for 10 min at 4°C using a benchtop centrifuge (Eppendorf, USA). An aliquot of 50 µL of 162 supernatant from each pool was combined to generate a single superpool for downstream 163 metagenomic analysis. 164 165 Library preparation and next generation sequencing 166 From the generated superpool, an aliquot of 140 µL of the supernatant was used for viral RNA 167 extraction with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) and eluted in a 168 single step with 60 µL of elution buffer, according to the manufacturer’s instructions. Paired -169 end sequencing libraries were prepared using the Illumina RNA Prep with Enrichment (L) 170 Tagmentation (Illumina, USA) following the manufacturer’s recommended protocol. The final 171 libraries were denatured with NaOH, diluted to a final concentration of 12 pM, and loaded onto 172 an Illumina MiSeq platform. Sequencing was performed using the MiSeq Reagent Kit v3 173 (Illumina, USA) to generate 300-bp paired-end reads. 174 175 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Sequence analysis and virus identification 176 Initial analysis was performed using the CZ -ID platform, an integrated pipeline that offers 177 quality control, de-hosting, duplicate removal, assembly, and viral identification capabilities. 178 After this initial processing, the de -hosted sequence reads were retrieved for further analysis. 179 To validate CZ-ID pipeline results, PrinseqLite v0.20.4 tool was used to filter low-quality reads 180 and remove adapters on command line. De novo sequence assembly was conducted using 181 MEGAHIT v1.2.9 (Li et al., 2015) where West Nile virus contigs were recovered and 182 compared to CZ -ID pipeline results. Only contigs with an average depth of coverage of ≥10 183 and a length of ≥500 bp were retained for further analysis. These contigs were first compared 184 against a local version of NCBI viral database using Diamond v2.0.4. To ensure specificity, 185 putative viral contigs were further compared to the entire non-redundant protein database (nr), 186 to exclude any non -viral contigs. A stringent e -value threshold of 1e -5 was employed 187 throughout the homology searches to minimize false-positive hits. 188 Phylogenetic analysis of the identified RNA viruses 189 To describe the identified viruses in an evolutionary context, publicly available viruses 190 belonging to these different groups, and more specifically those closely related to the viral 191 strains obtained in the current study were downloaded and used as reference sequences in the 192 reconstruction of phylogenetic trees. Closely related gene sequences were retrieved from NCBI 193 viral database and used as reference sequences in reconstructing the phylogenetic relationship 194 of the viral sequences. The combined set of sequences were aligned using MUSCLE software 195 with default parameters (max iterations = 16) embedded in Molecular Evolutionary Genetics 196 Analysis v.7.0 (MEGA7) (Kumar et al., 2016) platform. The aligned sequences were edited 197 using the Bioedit tool and maximum likelihood phylogenetic analysis carried out using IQ -198 TREE v1.6.12. The best model ( GTR+G4 (General Time Reversible + Gamma)) and tree 199 search was performed simultaneously based on 1000 bootstrap estimates and approximate 200 likelihood ratio test (aLRT). Genome maps were generated and visualized using Python v3.9. 201 202 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint

203 Mosquito Collection and Sequencing Output 204 Adult Aedes aegypti mosquitoes collected from Kwale County, coastal Kenya, were processed 205 for metagenomic analysis. Sequencing on the Illumina MiSeq platform generated 233,658 206 paired-end reads. After quality filtering, host read subtraction, and duplicate removal, 172,556 207 high-quality reads were retained for downstream virome analyse s. Insect-specific viruses 208 (ISVs) were detected exclusively in the Kwale County superpool. Superpools from Kilifi 209 (Malindi) and Mombasa were also processed and sequenced, however, no sequencing reads 210 were recovered, and consequently no insect-specific viruses or human-pathogenic arboviruses 211 were detected. 212 Metagenomic analysis 213 Metagenomic analysis revealed a diverse assemblage of insect -specific viruses (ISVs) 214 associated with Ae. aegypti mosquitoes from the study area. Viral contigs were assigned to 215 multiple taxonomic groups, representing both segmented and non-segmented RNA viruses. In 216 total, five ISVs were identified, including Fako virus (FAKV), Tesano Aedes virus (TEAV), 217 Aedes partiti -like virus (AePLV), Cell fusing agent virus (CFAV), and Formosus virus 218 (FORV). Complete genome sequences were recovered for Fako virus and Tesano Aedes virus, 219 while partial genome sequences were obtained for Aedes partiti -like virus, Cell fusing agent 220 virus, and Formosus virus. Genomic features of the detected viruses, including genome length, 221 GC content, closest reference sequences, and nucleotide identity, are summarized in Table 1. 222 Table 1: Viruses identified in this study based on metagenomic analysis. The identification 223 was carried out using a homology search against reference databases, providing insights into 224 the closest known virus. 225 226 227 228 229 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Strain Length GC Conten t % Closest hit Genbank ID % identity KWL_2022_AePLV 1,383 48.95 Aedes partiti-like virus- RdRp PV730257.1 99.49 1,349 48.04 Aedes partiti-like virus- Capsid OQ305266.1 99.26 KWL_2022_FORV 10,146 44.41 Formosus virus PV730233.1 99.47 KWL_2022_CFAV 2290 50.10 Cell fusing agent virus OQ305237.1 95.83 KWL_2022_TEAV 9,640 39.02 Tesano Aedes Virus PV658504.1 91.31 KWL_2022_FAKV 3,802 31.80 Fako virus -Segment 1 OR270150.1 98.73 3,735 32.88 Fako virus -Segment 2 OR270151.1 99.06 3,849 33.78 Fako virus -Segment 3 OR270152.1 99.19 3,385 31.23 Fako virus -Segment 4 OR270153.1 98.76 3,189 32.52 Fako virus -Segment 5 OR270154.1 97.70 1,747 34.29 Fako virus -Segment 6 OR270155.1 98.50% 1,149 36.81 Fako virus -Segment 7 OR270156.1 99.09% 1,131 37.05 Fako virus -Segment 8 OR270157.1 97.87% 1,274 32.34 Fako virus -Segment 9 OR270158.1 98.06% 230 231 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Phylogenetic analysis 232 Phylogenetic analyses based on the RNA -dependent RNA polymerase (RdRp) coding region 233 were conducted to determine the evolutionary relationships of the detected ISVs. Maximum 234 likelihood trees revealed that all identified viruses clustered within well -defined ISV clades, 235 consistent with their taxonomic classifications. Aedes partiti-like virus was detected as two 236 genomic segments corresponding to the RNA-dependent RNA polymerase (RdRp) and capsid 237 regions. Both segments exhibited high nucleotide identity (>99%) to known AePLV reference 238 sequences. 239 240 Figure 2. Maximum Likelihood phylogenetic tree of Aedes partiti-like virus (AePLV) inferred 241 from nucleotide sequences, with branch support assessed using 1,000 bootstrap replicates. 242 243 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Genome organization of AePLV 244 The assembled genome of AePLV was determined to be bi -segmented, with lengths of 1,383 245 bp and 1,349 bp. Segment 1 encodes the RNA -dependent RNA polymerase (RdRp), whereas 246 Segment 2 encodes the capsid and envelope -like proteins characteristic of insect -specific 247 viruses. The genome maps reveal a linear organization for both segments, with conserved open 248 reading frames (ORFs) and predicted untranslated regions (UTRs) at the 5’ and 3’ termini. 249 250 Figure 3. Genome map of AePLV showing two segments of varying lengths, each encoding a 251 separate open reading frame. 252 253 254 255 256 257 258 259 260 261 262 263 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Phylogenetic analysis of Formosus virus (FORV) showed that the recovered genome was 264 10,146 nucleotides in length with a GC content of 44.41%. The sequence exhibited high 265 nucleotide similarity (99.47%) to the closest reference strain (PV730233.1) and clustered with 266 a Nigerian strain, supported by a bootstrap value of 100. 267 268 Figure 4. Maximum Likelihood phylogenetic tree of Formosus virus (FORV) inferred from 269 nucleotide sequences, with branch support assessed using 1,000 bootstrap replicates. 270 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint The assembled genome of FORV consists of a single, continuous RNA segment of 10,146 bp. 271 The genome encodes a complete RNA -dependent RNA polymerase (RdRp) and several 272 structural proteins, including the capsid and putative envelope -associated proteins typical of 273 insect-specific viruses. Analysis of the genome map revealed a linear organization with clearly 274 defined open reading frames (ORFs) and untranslated regions (UTRs) at both the 5’ and 3’ 275 ends. 276 277 Figure 5. Genome map of Formosus virus (FORV) showing a single open reading frame in a 278 genome of 10,146 nucleotides. 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Phylogenetic analysis revealed that the Tesano Aedes virus genome clustered with a Kisumu -296 derived strain with a bootstrap value of 99. The genome showed nucleotide identity to the 297 closest available reference i ndicating notable genetic divergence from previously reported 298 strains. 299 300 Figure 6 . Maximum Likelihood phylogenetic tree of Tesano Aedes virus inferred from 301 nucleotide sequences, with branch support assessed using 1,000 bootstrap replicates. 302 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint The Tesano Aedes virus genome recovered in this study was 9,640 nucleotides in length and 303 contained a single open reading frame (ORF), characteristic of Iflaviruses, encoding a 304 polyprotein that is post-translationally processed into structural and non-structural proteins. 305 306 307 Figure 7. Genome map of Tesano Aedes virus (TEAV) depicting a single open reading frame 308 in a genome of 9,640 nucleotides, characteristic of Iflaviruses. 309 310 311 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Phylogenetic analysis of Fako virus revealed that the recovered genome clustered with a 312 previously reported Kenyan strain, indicating close genetic relatedness. The branching was 313 well supported in the phylogenetic tree, consistent with the virus circulating locally. 314 315 Figure 8. Maximum Likelihood phylogenetic tree of Fako virus (FAKV) inferred from RNA-316 dependent RNA polymerase (RdRp) gene sequences, with branch support assessed using 1,000 317 bootstrap replicates. 318 319 320 321 322 323 324 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint The Fako virus genome recovered in this study is segmented, comprising nine distinct segments 325 of varying lengths. Each segment encodes a separate open reading frame, with the RNA -326 dependent RNA polymerase (RdRp) segment used for phylogenetic analysis. This segmented 327 genome organization is consistent with previously described Fako virus strains and other 328 related segmented viruses. 329 330 331 332 333 334 335 Figure 9. Genome map of Fako virus (FAKV) showing nine segments of varying lengths, with 336 each segment encoding a separate open reading frame. 337 338 339 340 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Phylogenetic analysis of Cell Fusing Agent Virus (CFAV) revealed that the recovered genome 341 clustered with CFAV strains previously reported from Africa, indicating close genetic 342 relatedness across the continent. The branching in the phylogenetic tree was strongly 343 supported, reflecting the conserved nature of this insect-specific flavivirus. 344 345 Figure 10. Maximum likelihood phylogenetic tree of Cell Fusing Agent Virus (CFAV). 346 Bootstrap values were calculated from 1000 replicates. 347 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint

348 The virome of Aedes aegypti is increasingly recognized as being dominated by insect-specific 349 viruses (ISVs), which are unable to infect vertebrates due to their strict host tropism yet may 350 influence mosquito biology and arbovirus transmission dynamics. ISVs are found across 351 multiple viral families, including Flavivirida e, Iflaviridae, Partitiviridae and Reoviridae 352 reflecting both deep evolutionary relationships and potentially distinct ecological roles within 353 mosquito hosts. This study adds to the growing evidence that ISVs constitute a core component 354 of Ae. aegypti viromes and highlights the need to understand their broader biological and 355 epidemiological significance. 356 The detection of Aedes partiti -like virus 1 (AePLV) expands the growing evidence that 357 partitiviruses are stable components of mosquito viromes. Members of the family Partitiviridae 358 are double -stranded RNA viruses with typically bisegmented genomes encoding the RNA -359 dependent RNA polymerase (RdRp) and capsid protein (Li et al., 2023; Xia et al., 2018). 360 Although historically associated with plants and fungi, partiti -like viruses are increasingly 361 reported in insects, particularly mosquitoes, suggesting long -term host adaptation. Their 362 frequent detection across diverse geographic regions and mosquito populations implies 363 persistent infections, potentially maintained through vertical transmission. While their 364 functional role in mosquitoes remains unclear, recent studies suggest that persistent ISV 365 infections may influence host immunity and vector competence by modulating antiviral 366 pathways. 367 368 Formosus virus (FORV) and Tesano Aedes Virus (TeAV) belong to the family Iflaviridae, a 369 group of positive -sense single -stranded RNA viruses widely distributed among insects. 370 Iflaviruses typically establish persistent, often asymptomatic infections and are efficiently 371 maintained in mosquito populations. The repeated detection of TeAV in Aedes aegypti across 372 African regions supports its classification as a mosquito-adapted virus. Importantly, emerging 373 evidence indicates that TeAV may suppress dengue virus replication (Amoa-Bosompem et al., 374 2020), highlighting a possible indirect role in shaping arbovirus transmission dynamics. 375 Similarly, FORV contributes to the expanding diversity of mosquito -associated iflaviruses, 376 reinforcing the notion that iflaviruses are key constituents of the Aedes core virome with 377 potential ecological and epidemiological relevance. 378 379 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint The identification of Cell fusing agent virus (CFAV), a classical insect -specific flavivirus 380 (cISF), underscores the widespread circulation of insect -restricted flaviviruses in Aedes 381 populations. CFAV is evolutionarily distinct from mosquito-borne pathogenic flaviviruses but 382 shares genomic organization and replication strategies (Chiuya et al., 2021). Experimental 383 studies have demonstr ated that it can interfere with the replication of medically important 384 flaviviruses, including dengue and Zika viruses, through mechanisms such as superinfection 385 exclusion and immune priming (Martin et al., 2019) . Its presence in natural mosquito 386 populations therefore has important implications for arbovirus ecology, as co -infections with 387 ISFs may influence transmission efficiency and outbreak potential. 388 389 The detection of Fako virus (FAKV) places it within the family Reoviridae, a group of 390 segmented double-stranded RNA viruses known to infect a wide range of arthropods. FAKV 391 is characterized by a multi -segmented gen ome (nine segments ), consistent with mosquito -392 associated reoviruses (Auguste et al., 2014; Reinisch et al., 2000). Reoviruses are thought to 393 establish persistent infections and may be vertically transmitted, facilitating long -term 394 maintenance within vector populations. Although the biological impact of FAKV on mosquito 395 fitness and vector competence remains poorly understood, segmented ISVs such as FAKV 396 contribute substantially to virome complexity and may influence host antiviral responses 397 through continual immune stimulation. 398 399 The coexistence of insect -specific viruses from the families Partitiviridae, Iflaviridae, 400 Flaviviridae, and Reoviridae within Aedes aegypti underscores the taxonomic breadth and 401 ecological complexity of mosquito viromes. These viruses differ in genome organization and 402 replication strategy but share key features of persistent infection and host restriction. Increasing 403 evidence suggests that such insect-specific viruses are not neutral passengers; rather, they may 404 shape mosquito physiology and vector competence for arboviruses through immune 405 interactions, competitive exclusion, or modulation of cellular environments within the 406 mosquito host. Understanding these interactions is critical for interpreting arbovirus 407 surveillance data and could inform future virome-based strategies for arboviral disease control. 408 409

410 Collectively, these findings contribute to the growing understanding of mosquito -associated 411 viromes in Africa and emphasize the importance of incorporating insect -specific viruses into 412 arbovirus surveillance and vector biology studies. Characterizing ISVs at the family level 413 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint provides critical insight into their ecological roles and lays the groundwork for future 414 investigations into their potential application in biological control strategies and arbovirus 415 transmission mitigation. 416 417

418 This study was limited by viral detection being confined to Kwale County, despite processing 419 mosquito samples from Mombasa and Kilifi where no viruses were detected. The use of pooled, 420 cross-sectional metagenomic data limits inference on infection prevalence, viral replication 421 status, and seasonal dynamics. In addition, no arboviruses were detected, and functional 422 interactions between insect-specific viruses and arboviruses could therefore not be assessed in 423 this study. 424 425 Recommendations 426 • Expand spatial and longitudinal surveillance to capture seasonal and ecological 427 variation in ISV diversity. 428 • Conduct experimental studies to assess ISV–arbovirus interactions and their effects on 429 vector competence. 430 • Explore the potential of ISVs for biological control and paratransgenic interventions. 431 432 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint Funding 433 This work was funded by the Armed Forces Health Surveillance Branch (AFHSB) and its 434 Global Emerging Infections Surveillance (GEIS) Section, FY2022 ProMIS ID: P0116_22_KY 435 and FY2023 ProMIS ID P0094_23_KY. 436 Ethics approval and consent to participate 437 Ethical approval was obtained from the Kenya Medical Research Institute (KEMRI) Scientific 438 and Ethics Review Unit (SERU) under protocol number KEMRI/SERU/CCR/4702 and 439 WRAIR# 3101. Permission to conduct the study was granted by the National Council for 440 Science, Technology, and Innovation (NACOSTI). 441 Competing interests 442 The authors declare that they have no competing interests. 443

444 We thank, Victor Ofula, Dr. Samson Konongoi, Hellen Koka , Dr. Edith Chepkorir, Simon 445 Muhoro and Joseph Katur for their expert contribution in cell culture and data analysis. 446 447 Disclaimer 448 This Material has been reviewed by the Walter Reed Army Institute of Research. There is no 449 objection to its presentation and/or publication. The opinions or assertions contained herein 450 are the private views of the author, and are not to be construed as official, or as reflecting true 451 views of the Department of the Army or the Department of Defense. 452 Availability of data and materials 453 The sequences of the viruses identified in this study have been submitted to GenBank awaiting 454 accession numbers. 455 456 457 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 5, 2026. ; https://doi.org/10.64898/2026.01.05.697663doi: bioRxiv preprint

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