The La Crosse virus Gc head domain is a major determinant of infection and pathogenesis.

How orthobunyaviruses establish infections and disseminate to cause disease is not well understood. In a previous study using the in vivo evolution of La Crosse virus (LACV), we discovered a cluster of mutations localizing to the LACV Gc head domain. However, we do not understand how the Gc head domain contributes to infection. Here, we generated each of the aforementioned mutations and addressed the role of the Gc head domain in viral replication and infectivity in mammalian and insect cells. We found that specific head domain residues could attenuate replication and infectivity in human neurons and reduce cell binding across different hosts, indicating an important role for the head domain during infection in vitro. Focusing on the in vitro-attenuated Gc N609D variant, we infected 3-week-old wild-type (WT) C57BL/6J mice via the footpad with WT LACV or the Gc N609D variant and found that the Gc N609D virus was completely attenuated. To address whether the variant was also attenuated in a highly susceptible mouse model, we infected Ifnar1-/- mice with WT LACV and Gc N609D. We found that virulence in mice infected with Gc N609D was delayed, with several mice surviving the infection, and while viral titers were comparable between viruses in the footpad, there was a significant reduction in virus in the brain. Together, these studies define a critical role of the Gc head domain in infectivity and pathogenesis. Studies are underway to further define how the orthobunyavirus Gc head domain contributes to infection and disease.IMPORTANCEOrthobunyaviruses are emerging arboviruses capable of severe disease and explosive outbreaks. However, our understanding of how orthobunyaviruses establish infections or cause disease is not completely understood. The orthobunyavirus Gc glycoprotein contains a variable amino-terminal head domain that forms the tip of the virion trimeric spike, yet it is unclear how the head domain contributes to infection or pathogenesis. In this study, we use LACV and a panel of Gc head domain variants to address the role of the head domain in LACV biology. We found that critical head domain regions are important for virus infectivity and pathogenesis in mice, highlighting an important role for the Gc head domain in orthobunyavirus infection and disease.