La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, Aedes triseriatus and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. Our surveillance study demonstrated the involvement of an invasive species, Aedes japonicus, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. LACV infection did not affect the host-seeking behavior of Ae. triseriatus females. Using high performance liquid chromatography with electrochemical detection (HPLC-ED), the levels of serotonin and dopamine were measured in infected and uninfected mosquitoes. Serotonin is known to affect blood-feeding and dopamine affects host-seeking. Serotonin levels were significantly lower in LACV-infected mosquitoes but dopamine levels were unaffected by virus. A previous study found that LACV infection caused an alteration in mosquito blood-feeding in a way that could enhance virus transmission. This work showed that LACV infection can reduce the level of serotonin in the mosquito, promoting virus transmission through altered blood-feeding without impairing the vector's ability to locate a host. Standard CDC bottle assays were used to evaluate the efficacy of two pyrethroids and two essential oil sprays on LACV infected and uninfected mosquitoes. LACV-infected Ae. triseriatus females were more susceptible to both pyrethroids than uninfected ones. Infection status did not affect the susceptibility of Ae. albopictus to either pyrethroid. The essential oils were inconsistent in their effects. These results demonstrate that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virus-infected portion of the vector population. / Ph. D. / La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, <i>Aedes triseriatus</i> and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. The surveillance study demonstrated the involvement of an invasive species, <i>Aedes japonicus</i>, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. Our study of the effects of LACV infection on host-seeking and neurotransmitter levels showed that LACV can manipulate <i>Ae. triseriatus</i> females in a way that could facilitate transmission of the virus. Lastly, we showed that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virusinfected portion of the vector population.

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. / Doctor of Philosophy / La Crosse virus (LACV) is a mosquito-borne virus that can cause severe illness in children under 16 years of age through the bite of an infected mosquito. Infected children can develop severe neuroinvasive disease, including encephalitis, seizures, coma, and death. Following recovery, some children experience continued effects (neurological sequelae) of the infection, such as epilepsy, hemiparesis (weakness on one side of the body), and neurobehavioral issues. 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 through transmission from mother to offspring in mosquitoes. Historically, the majority of LACV cases occurred in the Midwest, but recently, rising cases in the Appalachians are a growing concern. Cases in Appalachia now outnumber those in the Midwest and cause most cases in the US. Interestingly, 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. It is unclear why Virginia is not reporting as many LACV cases as other states, but it may simply be due to reduced circulation in the state. In chapter two, we investigate the circulation of LACV in Virginia by examining wildlife exposure to LACV. Blood samples from local wildlife rehabilitation centers (527 samples comprising 9 vertebrate species) were tested for the presence or absence of antibodies against LACV. We identified a seroprevalence rate of 1.9% in 5 vertebrate species. In addition to known vertebrate hosts of LACV (Eastern Gray Squirrel, Eastern Cottontail, and Groundhog), we identified the first known Red Fox exposed to LACV, potentially implicating it in the transmission cycle. In addition to the historic LACV lineages, a new lineage, lineage III, was isolated in the Northeast, and additional isolations have been made. However, there have not been any reported clinical cases attributed to lineage III. 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 ability of vector mosquitoes to transmit LACV lineage III both through an infected bite and from mother to offspring. We were able to show that lineage III can be transmitted through an infected bite and from mother to offspring, and we highlight the potential of a public health risk for lineage III LACV. In chapter four, we explore the pathogenesis of LACV lineage III in an immune-competent and immune-deficient mouse model. Lineage III was able to cause severe disease similar to that in children in the immune-deficient model but not in the immune-competent model. Historic LACV lineages can overcome the host immune system through the antiviral interferon system, but our data indicate that lineage III may be unable to overcome this system and, thus, unable 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. However, viruses can evolve quickly, and we still advocate for continued surveillance and investigation into lineage III LACV.

Zika (ZIKV), La Crosse (LACV), and Cache Valley (CVV) viruses are mosquito-vectored diseases that cause significant morbidity and mortality in humans and animals. Transmission of these viruses are dependent on numerous factors including vector competence and the effects of mosquito-virus interactions. We conducted vector competence studies of local Aedes and Culex mosquitoes for ZIKV and CVV, and found that all Aedes mosquitoes were competent for CVV and only Aedes albopictus and Aedes japonicus were competent for ZIKV. Vector competence for CVV was dose-dependent, where mosquitoes orally infected with high titers developed higher transmission rates. We also found that vector competence for ZIKV was limited by midgut and salivary gland barriers. Second, we looked at the effects of LACV and ZIKV infection on repellent response in Aedes mosquitoes and found that infected mosquitoes were refractory to low concentrations of DEET, picaridin, and PMD. Increasing concentrations of the repellents to ≥10% was able to increase percent protection (%p) against infected and uninfected mosquitoes. Lastly, we determined the effects of ZIKV and LACV infection on oxidative stress and glutathione-S-transferase (GST) activity in Aedes albopictus. Virus infection had no effect on oxidative stress, but GST activity was significantly different for mosquitoes 3-days post-exposure. We found that oxidative stress levels and GST activity had an inverse relationship for infected and uninfected mosquitoes, where oxidative stress decreased and GST activity increased over the 10-day test period. This indicates that GSTs may aid in controlling byproducts of oxidative stress. The results from this entire study identified competent vectors for emerging arboviruses and demonstrated the behavioral and physiological effects of virus infection in the mosquito vector. / Doctor of Philosophy / Zika (ZIKV), La Crosse (LACV), and Cache Valley (CVV) viruses are transmitted by mosquitoes and can make humans and animals very sick. There are many biological factors that determine if a mosquito can transmit a virus and these viruses can change the biology of a mosquito. We conducted laboratory studies to see if Aedes and Culex mosquitoes can transmit ZIKV and CVV. We found that all Aedes mosquitoes were able to transmit CVV and only the Asian tiger mosquito and Asian rock pool mosquito were able to transmit ZIKV. Mosquitoes infected with high amounts of CVV developed higher transmission rates. We also found that transmission of ZIKV was limited by barriers in the mosquito midgut and salivary glands. Second, we looked at the effects of LACV and ZIKV infection on how Aedes mosquitoes respond to repellents and found that infected mosquitoes were less sensitive to low concentrations of DEET, picaridin, and PMD. Increasing concentrations of the repellents to 10% or higher was able to provide adequate protection against infected and uninfected mosquitoes. Lastly, we determined the effects of ZIKV and LACV infection on oxidative stress and glutathione-S-transferase (GST) activity in the Asian tiger mosquito. Virus infection did not change oxidative stress, but GST activity was higher in infected mosquitoes tested after 3 days after infection. We found that oxidative stress decreased and GST activity increased over the 10-day test period. This indicates that GSTs may help control damaging products from oxidative stress. The results from this entire study identified what mosquitoes were able to transmit emerging mosquito-borne viruses and demonstrated the biological effects of virus infection in the mosquitoes.