Jamestown Canyon Virus Rapidly Adapts to Mosquito Cells Through Multiple M Segment Mutations

Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus with a broad host and vector range. Despite increasing mosquito-to-human spillover, the viral determinants governing host adaptation remain poorly defined. We examined changes in JCV replication during serial passage in mosquito cells and linked adaptive changes in viral fitness to specific genetic mutations. In mosquito-derived C6/36 cells, JCV exhibited a distinct "lag-burst" phenotype in which viral replication remained nearly undetectable for 10 days before abruptly increasing. Following reinfection of fresh C6/36 cells, JCV passaged once in mosquito cells exhibited robust replication with no detectable lag phase. Sequencing identified seven substitutions compared to the published reference sequence, including S193G in Gn, three NSm substitutions (P346A, M352V, and W371R), and three Gc substitutions (H890Y, I1365V, and L1394F). Using a plasmid-based reverse genetics system, individual mutations were introduced into recombinant JCV and tested for effects on mosquito cell replication. All tested mutations independently enhanced replication efficiency, demonstrating that adaptation can arise through multiple independent genetic pathways. However, no individual mutation fully reproduced the phenotype acquired naturally through mosquito cell passage. Together, these findings demonstrate that JCV rapidly adapts to mosquito cells under minimal selective pressure and highlight the potential for emergence of increasingly well-adapted viral variants.