A Novel Mycovirus Reprograms the Pathogenic State of Aspergillus flavus in Keratitis

Purpose: Fungal keratitis (FK) is a major cause of corneal blindness, with substantial variability in disease severity that cannot be fully explained by host factors. This study investigated whether endogenous mycovirus infection contributes to differences in fungal pathogenicity. Methods: Fungal isolates from 38 patients with FK were analyzed by metagenomic sequencing. A novel viral RNA identified in an Aspergillus flavus isolate was characterized by sequence and phylogenetic analyses. Virus-cured strains were generated and compared with virus-infected strains using phenotypic assays, transcriptomic profiling, corneal epithelial adhesion assays, macrophage killing assays, and a murine model of FK. Results: A previously uncharacterized mycovirus, A flavus narnavirus 3, was identified within the family Narnaviridae. Viral infection impaired fungal growth, conidiation, and stress tolerance and suppressed virulence-associated traits, including sclerotia formation and aflatoxin production. A transcriptomic analysis revealed the disruption of the regulatory networks involved in development, stress responses, and metabolism. Functionally, virus-infected strains showed reduced adhesion to corneal epithelial cells and increased susceptibility to macrophage-mediated clearance. In vivo, A flavus narnavirus 3-positive strains exhibited attenuated virulence, with a lower fungal burden and reduced corneal inflammation. Conclusions: Mycovirus infection attenuates A flavus pathogenicity and modulates host responses in FK, suggesting a potential role for viral carriage in the clinical heterogeneity of disease severity.