Usutu virus (USUV) is an emerging zoonotic virus within the Flaviviridae family that can cause neurological disease in humans and wild birds. USUV is maintained in an enzootic cycle between wild birds, primarily passerine species, and ornithophilic mosquitoes, predominantly Culex spp. mosquitoes. Since its first isolation in 1959 in South Africa, USUV has spread throughout sub-Saharan Africa and Europe. Its emergence into Europe was marked by large die-offs, or epizootics, of the Eurasian blackbird (Turdus merula), as well as an increase in human cases. This dissertation sought to understand whether USUV has evolved to become more pathogenic in humans or transmissible in birds. We compared the pathogenesis of five different USUV isolates, four recent isolates: Spain 2009, Netherlands 2016, Senegal 2003, Uganda 2012, and South Africa 1959, in an interferon α/β receptor knockout (Ifnar-/-) mouse model. We observed significant mortality, high viral levels in serum and tissues in all USUV strains except for the Netherlands 2016 strain. Eighteen non-synonymous mutations were identified throughout the genome of Netherlands 2016 strain compared to the other USUV isolates. To further understand USUV infection in wild birds, we developed a physiologically relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more pathogenesis and higher viral titers in tissues compared to the African strains. This work established the first viremic bird model of USUV infection. Passerine birds have been suggested to be important for USUV maintenance, however a species competent for transmission has not been identified. We first determined that wild-caught house sparrows (Passer domesticus) and Culex quinquefasciatus mosquitoes were susceptible to Netherlands 2016 and Uganda 2012 USUV strains. Following an infectious feed to assess enzootic transmission, house sparrows were able to transmit both USUV strains to Cx. quinquefasciatus mosquitoes, with the Netherlands 2016 strain being more infectious compared to the Uganda 2012 strain. The collection of these chapters provides great insights on the pathogenesis of distinct USUV strains, disease presentation in birds, and enzootic transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is entirely feasible. / Ph.D. / Usutu virus (USUV) is a mosquito-borne virus that can cause neurological disease in humans and wild birds. It is in the same virus family as West Nile virus and St. Louis encephalitis virus, both of which already occur in the United States. USUV is typically maintained by spreading back and forth between wild birds, typically songbird species, and mosquitoes with an affinity for biting birds, such as Culex spp. mosquitoes. USUV was first found in South Africa in 1959 and has since spread throughout sub-Saharan Africa and Europe. Upon emergence in Europe, USUV caused massive die-offs in Eurasian blackbirds (Turdus merula) as well as an increase in human cases. My work sought to understand whether USUV has evolved to cause more disease in humans or transmissibility in wild birds. We first assessed the disease severity of African and European USUV strains in an immune compromised mouse model. We showed that African strains of USUV caused more disease and developed higher viral loads than European strains in mice that lack full immunity. To further understand USUV infection in wild birds, we developed a relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more disease and higher viral loads in tissues compared to the African strains. This work established the first bird model of USUV infection where birds developed infectious virus that is detectable in blood. Finally, to identify possible reservoir hosts of USUV, we assessed USUV transmission between house sparrows (Passer domesticus) and Cx. quinquefasciatus mosquitoes. We found that house sparrows were able to transmit a European and African USUV strain to biting Cx. quinquefasciatus mosquitoes, with the European strain being more infectious to these mosquitoes. This indicates that house sparrows are competent hosts for USUV transmission and thus a potential reservoir species. The collection of these chapters provides great insights on the severity of distinct USUV strains, disease presentation in birds, and transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is a very real possibility.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / Japanese encephalitis virus (JEV) is a virus of the Flavivirus genus that may result in encephalitis in vertebrate hosts. This vector-borne zoonosis occurs in Eastern and Southeastern Asia and an intentional or inadvertent introduction into the United States (US) would lead to important public health and economic consequences. The objective of this study was to gather, appraise, and synthesize primary research literature to identify and quantify vector and host competence for JEV, using a systematic review-metaanalysis (SR-MA) approach. After defining the research question, we performed a search in selected electronic databases. The title and abstract of the identified articles were screened for relevance using a defined set of exclusion and inclusion criteria, and relevant articles were subjected to a risk of bias assessment followed by data extraction. Random-effects subgroup meta-analysis models were fitted by species (mosquito or vertebrate host species) to estimate pooled summary measures as well as to compute the variance between studies. Meta-regression models were fitted to assess the association between different predictors and the outcomes of interest and to identify sources of heterogeneity among studies. Data were extracted from 171 peer-reviewed articles. Most studies were observational (59.06%) and reported vector competence (60.2%). The outcome measures reported pertained to transmission efficiency, host preference, and vector susceptibility to infection within vector competence; and susceptibility to infection within host competence. All outcome measures (JEV proportion of infection in vectors and hosts from observational studies; and JEV infection, dissemination, and transmission rates in vectors from experimental studies) had high heterogeneity. Mosquito species, diagnostic method, country, and capture method represented important sources of heterogeneity associated with the proportion of JEV infection in vectors; host species and region were considered sources of heterogeneity associated with the proportion of JEV infection in hosts; and diagnostic and mosquito capture methods were deemed important contributors of heterogeneity for the minimum infection rate (MIR) outcome. Mosquito species and administration route represented the main sources of heterogeneity associated with JEV infection rate in vectors. Quantitative estimates resulting from this SR-MA will be inputted into risk assessment models to evaluate risks associated with the introduction of JEV in the US.