The La Crosse virus M segment determines virus isolate cell-to-cell spread and virulence

La Crosse virus (LACV) is an orthobunyavirus spread by mosquitoes in North America and can cause severe neurological disease. Despite this burden, there is a lack of LACV antiviral treatments as our fundamental understanding of how LACV spreads and causes disease remains incomplete. To investigate LACV biology, we took advantage of two genetically similar LACV lineage I isolates (LACV₁₉₆₀ and LACV₁₉₇₈) where we found that LACV₁₉₆₀ infects and replicates at a higher rate than LACV₁₉₇₈, while LACV₁₉₇₈ exhibits enhanced cell-cell spread and in vivo virulence and dissemination. To investigate the genomic determinants behind these phenotypes, we generated reassortants between each isolate. We found that each genomic segment contributed to LACV pathogenesis with the M segment as a dominant determinant for LACV pathogenesis in vivo and plaque size and replication in vitro. To address which M segment protein contributes to plaque size and infectivity, we generated M segment chimeric viruses using a LACV₁₉₇₈ background and swapping in regions of the LACV₁₉₆₀ M segment. We found that the Gc head domain determined plaque size and infectivity, with the LACV₁₉₆₀ Gc head chimera producing small plaques but having increased infectivity over the wild-type LACV₁₉₇₈. Finally, using natural LACV lineage isolates, we showed that plaque size is variable across and within lineages suggesting changes in the M segment may impact LACV in nature. In future studies, we will continue to investigate the mechanisms behind how cell-to-cell spread influences dissemination to better understand how LACV genome segments affect spread, infectivity, virulence, and viral fitness.