(1) Mechanisms of virulence acquisition in Ngari virus (NRIV): In 1997-8 a large outbreak of Rift Valley fever (RVF) occurred in Kenya and Somalia. During this outbreak, NRIV was identified as the causative agent of hemorrhagic fever (HF) in a significant proportion of the cases. NRIV has been recognized as a naturally occurring genetic reassortant between Bunyamwera virus (BUNV; L and S segments) and Batai virus (BATV; M segment), both of which belong to the Bunyamwera serogroup in the genus Orthobunyavirus. Interestingly, both parental viruses cause febrile illness, but not severe HF in humans. This is a perfect example of the important role that genetic reassortment plays in the evolution of viruses and of the changes in virulence that can result. Therefore, we are using NRIV as a model to understand the molecular mechanisms underlying the emergence of novel pathogenic bunyaviruses in nature. To identify the molecular determinants responsible for the acquisition of virulence by NRIV, we first determined the full-length genome sequences of five NRIV strains, including the human isolates responsible for the HF outbreaks in Kenya and Somalia, as well as five BATV strains from diverse geographical locations. Our current studies in this regard are now focusing on the comparison between BUNV and BATV with regards to biological properties that might contribute to the heightened virulence of the reassortant associated with NRIV genotype. We have examined receptor usage, which could potentially affect the ability of these viruses to infect different cell subsets; however, we found that receptor usage seems to overlap between these two virus groups. We have also explored the possibility that growth kinetics and the robustness of replication in target cells may contribute to differences in virulence. We have found apparent difference in the susceptibility of macrophages to infection with BUNV, BATV, and NRIV. Among the three viruses, NRIV grew to the highest titers in primary human macrophages, while there were no obvious differences in the growth of these three viruses in mosquito cells, suggesting that NRIV specifically acquired an enhanced ability to grow in human macrophages during reassortment. In addition, we reveled that BUNV, BATV, and NRIV induce different inflammatory response profiles in human primary macrophages. (2) Genetic characterization and evolutionary modelling for orthobunyaviruses and uncharacterized bunyaviruses: The Bunyaviridae is an unusually large and diverse virus group, with the genus Orthobunyavirus alone containing more than 150 named viruses, among which are numerous human pathogens. In the absence of a sufficient body of genetic data to facilitate molecular identification, orthobunyaviruses have historically been classified into one of 18 distinct serogroups, again reflecting the high degree of genetic diversity among these viruses. However, this approach has significant limitations in that it usually focuses on only one or at most two viral antigens, making it much less likely that reassortants will be identified using this approach, particularly in the case of reassortants within a serogroup. In addition, serological cross-reactivity can be high among certain viruses, preventing a definitive identification based on such methods. Alternatively, some uncharacterized bunyaviruses do not show sufficient reactivity to any known group and as a result their exact nature remains unknown. However, these viruses are particularly important to our understanding of the genetic diversity that exists in nature and may also provide critical links in our understanding of the evolution of this group. In order to address these gaps in our knowledge and generate datasets of sufficient size and completeness for evolutionary analysis, we have undertaken large-scale sequencing of various orthobunyavirus groups, including the Bunyamwera, Simbu, Bwamba/Pongola, Nyando, and other uncharacterized group viruses in the genus Orthobunyavirus, which are prevalent but understudied causes of febrile illness worldwide. Among them, Guaroa virus (GROV) is a frequent but understudied cause of febrile illness throughout South America where it is transmitted by mosquitoes. Despite its public health importance, until now there has been little data available for assessing the genetic diversity among GROV isolates or modelling virus evolution and spread. To address this, complete sequencing of 12 geographically and temporally diverse isolates of GROV was undertaken. Analysis of sequence divergence combined with phylogenetic analysis showed that, with the exception of an early Brazilian isolate (strain BeH22063), for which the only pre-existing sequence data was available, all other isolates were closely related. In addition, the close relationship of GROV to the Wyeomyia group viruses (WYOV) allowed us to model virus spread following the introduction of their common ancestor into the Central/South American region. These analyses suggest that the spread of GROV into Peru and Bolivia, where they currently pose a significant problem, is most likely a recent event and that careful monitoring for further expansion of the endemic region is warranted. (3) Molecular characterization of tick-borne phleboviruses potentially causing human disease: In order to better understand the relationships between the molecular biological characteristics of uncharacterized viruses and their zoonotic potential, as well as their evolution, we are conducting an extensive genetic analysis and biological characterization of uncharacterized taxonomically ungrouped bunyaviruses associated with ticks, including those isolated from Africa, Asia, and South and North America. During 2005-2013, Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) and Heartland virus, two novel tick-borne phleboviruses, were first recognized as the causes of severe illness with thrombocytopenia among humans in China, Japan, and South Korea, or the United States, respectively. Although these tick-borne phleboviruses (TBPVs) comprise a related group in the genus Phlebovirus, along with the Bhanja group viruses (BHAVs) and Uukuniemi group viruses (UUKVs), the epidemiological study and diagnosis of all TBPVs simultaneously has been difficult due to the high degree of serological and genetic divergence among these viruses. Therefore, based on available TBPV genome sequences, determined by us and others, we have developed an RT-PCR system targeting a conserved sequence motif in the L genome segment that is able to detect the L segment RNA of all known TBPVs. This RT-PCR assay allowed us to identify some taxonomically unassigned bunya-like viruses as TBPVs. Furthermore, by collaborating with Hokkaido University and Yamaguchi University in Japan, we have discovered novel TBPVs in Japan, Mali, Zambia, and the United States using our RT-PCR system. Phylogenetic analysis revealed that viruses associated with ticks are more divergent than was previously appreciated and represent the dominant virus type within the genus Phlebovirus, as compared with mosquito/phlebotomus-borne viruses (e.g., Rift Valley fever virus). From these tick samples, we succeeded in isolating a novel tick-associated phlebovirus, Mukawa virus (MUKV), from Ixodes persulcatus in Japan. Full-length genome analyses on MUKV demonstrated that this virus may genetically share a common ancestor with mosquito/sandfly-borne phleboviruses. In vitro characterization of this virus revealed that MUKV leads to a productive infection in human cell lines and tick cells but not in mosquito cells, suggesting that MUKV is a TBPV despite its genetic similarities to the mosquito-borne viruses. This study suggests that MUKV is a novel and unique TBPV, and it also facilitates a better understanding of phlebovirus evolution and their vector range determinants.