Single-cell Characterization of Skin Response to a Bite by West Nile Virus-Infected Mosquito Reveals Fibroblast-Mediated Barrier to Transmission

Cutaneous events determining transmission of mosquito-borne orthoflaviviruses remain largely uncharacterized. Here, we report single-cell RNA-sequencing of skin from immunocompetent mice exposed to West Nile virus-infected mosquitoes, capturing early response at a critical transmission bottleneck. Skin-resident fibroblasts, keratinocytes, and myeloid cells were exposed to infectious saliva. At the bite site, neutrophils and mast cells diminished, while lymphoid cells augmented. Cell-cell communication analysis showed that structural skin cells activate immune signaling targeting myeloid populations, which signal to lymphoid cells. Transcriptional profiling revealed cell subtype-specific responses integrating immune activation, skin repair and metabolic remodeling. Using in vivo gene silencing, we demonstrated that fibroblast-expressed LRRC15 (leucine rich repeat-containing 15) functions as a restriction factor, limiting viral replication in skin, viral dissemination to draining lymph nodes, and disease severity. Collectively, our transcriptomic-resolution and functional analyses provide cellular and molecular understanding of bite-initiated arboviral transmission, establishing skin-resident fibroblasts as frontline defender cells. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest. Fondation pour la Recherche Médicale, SPF202110013925, ARF202309017577 Agence Nationale de la Recherche, ANR-20-CE15-0006, ANR-21-CE15-0041 EU Horizon, 101137006 PHC Siam, 49487UJ CIRM, EDUC4-12822