Abstract
Discovering divergent RNA viruses remains challenging due to the limitations of reference-based sequencing and viral isolation. Here, we present the structure-guided discovery of Aedes albopictus-associated Tymoviridae-like virus strain IMCAS (AaTLV-IMCAS), isolated from C6/36 mosquito cell supernatant. Without prior genomic information, we determined its capsid structure at a resolution of 2.11 Å using cryogenic electron microscopy (cryo-EM). Deep-learning-based automated modeling via ModelAngelo generated position-specific residue profiles that directed the targeted capture and assembly of the complete 6,488-nucleotide positive-sense single-stranded RNA genome, establishing a structural discovery workflow named CryoSeekV. Phylogenetic analysis showed that AaTLV-IMCAS, along with the related mosquito-borne viruses, forms an independent monophyletic clade distinct from established plant-infecting genera, representing a novel genus; therefore, we proposed the genus Mosquivirus, within the family Tymoviridae. Structural analysis revealed a icosahedral capsid where a 9 Å main-chain shift within the N-terminus acts as a conformational switch governing quasi-equivalent capsomer assembly. Functional assays demonstrated a strict mosquito cell tropism and temperature-dependent replication optimized at 28 °C that establishes persistent infection without cytopathic effects. Time-resolved transcriptomics revealed that infection triggers dynamic host reprogramming: an early (24 h) surge in host translation, oxidative phosphorylation, and amino-acid metabolism coupled with selective repression of key melanization and recognition factors, which largely returned toward baseline by 48 h. Together, this study establishes CryoSeekV as an effective paradigm for structure-driven viral surveillance and provides foundational molecular and physiological insights into invertebrate-associated Tymovirales.