Mosquito-borne viruses, including dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), are rapidly emerging, global pathogens. Though the number of people impacted by each virus varies, there have been thousands to millions of people infected. The focus of this thesis work centers around USUV and WNV; both have RNA genomes and belong to the Flaviviridae virus family. Both WNV and USUV were initially isolated in Africa and have since spread to Europe; interestingly, WNV has also spread globally and is considered endemic in the Americas. Similar to other flaviviruses, USUV and WNV are maintained in a mosquito vector-avian host transmission cycle, with spillover infection into humans. Human infections of WNV and USUV are usually asymptomatic, but in severe cases can cause neuroinvasive disease. WNV and USUV belong to the JEV serocomplex group, which indicates that antibodies produced against these viruses share a common antigen; the common antigen is hypothesized to be the envelope (E) protein on the outside of the virion. Neutralizing antibodies against both WNV and USUV have been found in birds and humans across Europe. In vitro cross-neutralization of WNV and USUV has been modeled experimentally and been observed in clinical settings. The neutralizing antibody response generated against WNV has been studied extensively in mouse models; however, there are few studies which examine the neutralizing antibody response generated against USUV. Whether prior WNV exposure protects against USUV disease is also unknown. The main goal of this thesis was to characterize how a primary flavivirus exposure would influence a secondary flavivirus exposure; specifically, we wanted to observe if WNV exposure would protect against USUV disease in vivo and generate a cross-neutralizing antibody response in vitro. For the WNV exposure, we used an attenuated vaccine strain of WNV that contains the WNV E gene (D2/WN-V3) developed by our collaborators. We hypothesized that treatment with D2/WN-V3 would protect against USUV infection. Two in vivo models were used: CD-1 mice and interferon alpha-beta receptor 1 deficient (Ifnar1-/-) mice. We discovered that sera from mice vaccinated with D2/WN-V3 neutralized both WNV and USUV in vitro. In the Ifnar1-/- model, we observed that vaccinated mice had higher survival rates and lower USUV viremia levels after USUV challenge. This work helps characterize the consequences of flavivirus antibody cross-neutralization in vitro and cross-protection in vivo. As the flavivirus field moves toward the goal of creating a pan-flavivirus vaccine, both cross-reactive antibodies and cross-protection need to be considered. / Master of Science / West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that were originally isolated in Africa during the 20th century. Both viruses are maintained through a transmission cycle between mosquito vectors and avian hosts. Mosquitos transfer the infectious agent (WNV or USUV) through feeding on a bird (usually a passerine species); once in the bird, the virus can replicate to high levels. Human infections of WNV and USUV from mosquitos can also occur, with symptoms ranging from mild febrile illness to severe encephalitis or meningitis. Over the past few decades, WNV and USUV have spread to Europe, most likely through infected migratory birds. Interestingly, mosquito surveillance studies in mainland Europe have found mosquitos that tested positive for both USUV and WNV. In Europe, antibodies for both viruses have been found in humans and birds, indicating a previous exposure to WNV, USUV, or both. The neutralizing antibody response is a critical immune defense against viral infections. Neutralizing antibodies bind strongly to the outside of the virion (virus particle), preventing the virion from interacting with and infecting the host cell. For WNV and USUV, one of the targets that neutralizing antibodies bind to is the outer envelope (E) protein of the virion. In clinical settings and experimental studies, cross-neutralization of WNV and USUV has been documented. During cross-neutralization, a serum sample containing neutralizing antibodies against WNV can also neutralize USUV, and vice versa. Although the neutralizing response against WNV has been characterized in humans and lab animal models such as mice, there is little research regarding the neutralizing response against USUV. Importantly, whether prior WNV exposure provides protection against USUV infection is currently unknown. The main goal of this thesis was to characterize the disease outcome and neutralizing response against USUV after a WNV exposure. For the WNV exposure, we used a vaccine strain of WNV that contains the E gene (D2/WN-V3) developed by our collaborators. We predicted that vaccinated mice would avoid USUV clinical signs of disease and generate neutralizing responses to WNV and USUV. To do this work, we used two laboratory mouse models: mice with an intact immune response system (CD-1) and mice with a stunted immune response (Ifnar1-/-). We discovered that serum from vaccinated mice did cross-neutralize WNV and USUV. In the Ifnar1-/- model, vaccinated mice had higher survival rates and lower levels of virus in blood after USUV infection compared to unvaccinated mice. Ultimately, this work highlights the importance of characterizing the immune response against similar viruses and will inform the development of human vaccines for both viruses.

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