Comparative Transmission and Pathogenesis of La Crosse Virus (LACV) Lineages and Evidence for LACV Circulation in Virginia Wildlife

La Crosse virus (LACV) is a mosquito-borne arbovirus that is the main cause of pediatric encephalitis in the United States in children under 16. LACV is maintained in hardwood forests utilizing Aedes spp. mosquitoes, primarily Aedes triseriatus, and Sciuridae vertebrate host species, primarily the Eastern Chipmunk (Tamias striatus) or via transovarial transmission from mother mosquitoes to offspring. Historically, LACV comprised two genetically distinct lineages: lineage I in the Midwest and Appalachian regions and lineage II in the southern region. Lineage I LACV is a growing public health concern in Appalachia, where cases now outnumber those in the Midwest, which historically accounted for most LACV cases. The majority (53%) of LACV cases between 2003 and 2023 were reported from Appalachian states surrounding Virginia (North Carolina: 331, Tennessee: 213, West Virginia: 225, and Kentucky: 11), but Virginia only reported 25 cases. In chapter two, we explore the hypothesis that LACV is circulating in Virginia utilizing serosurveillance to indicate LACV circulation. Blood samples provided by wildlife rehabilitation centers throughout Virginia (n=527 comprising 9 vertebrate species) were screened for LACV antibodies using Plaque Reduction Neutralization tests (PRNTs). We identified an overall prevalence rate of 1.90% across five seropositive vertebrate species. In addition to known hosts of LACV, Eastern Gray Squirrel, Eastern Cottontail, and Groundhog, we identified the first reported seropositive Red Fox. We highlight that LACV is circulating in Virginia, although at a much lower rate than reported from other states, which may explain the reduced number of cases. In addition to lineage I and II LACV, a new lineage was identified in 2005 in the Northeast but has yet to cause human disease. In the face of an apparent entomological risk demonstrated by the continued isolation of lineage III from mosquitoes in this region, it is unclear why there is a lack of clinical cases. The prevailing hypotheses are (1) under-diagnosis in cases of human disease or lack of detection in humans, (2) reduced virulence in lineage III LACV, (3) low prevalence of lineage III in local mosquito and host animal populations, or (4) reduced vector competence in local mosquito populations. In chapter three, we explore the vector competence of Aedes albopictus and Aedes triseriatus from the range of lineage III (Connecticut) and from the historic LACV range (Virginia) to transmit LACV lineage III. Using oral feeding and intrathoracic inoculation, bodies, legs, saliva, and ovaries were harvested to indicate infection, dissemination, ability to transmit, and ability to vertically transmit, respectively. Although there were no apparent differences in horizontal or vertical transmission, we demonstrated that LACV lineage III can be transmitted both horizontally and vertically, highlighting the potential public health risk associated with lineage III. In chapter four, we explore the pathogenesis of lineage III. An immune-competent murine model, CD-1, and an immune-deficient murine model, IFNAR-/-, were used to determine the pathogenesis of lineage III. Interestingly, in the immune-deficient model, lineage III was able to cause significant morbidity and mortality, but not in the immune-competent model. LACV lineage I and II can overcome the host immune system through the interferon pathway, allowing viral replication. We hypothesize that lineage III cannot circumvent the interferon system as the other lineages can and, therefore, cannot replicate to cause clinical disease. Overall, although lineage III can be transmitted in vector mosquitoes, it may be unable to cause disease in humans because it cannot overcome the antiviral responses, but this needs to be explored further to determine the underlying mechanism. However, viruses can evolve quickly, and we still advocate for continued surveillance and investigation into lineage III LACV.