(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. We have recently determined the full genome sequences of 5 NRIV isolates (genotype: L-BUNV/M-BATV/S-BUNV), including 2 strains from the Kenya/Somalia HF outbreak, as well as 5 BATV isolates, including the UgMP-6830 strain, which is the closest relative of the NRIV M-segment, and 4 BUNV isolates, among which we have identified a recent field strain that appears to be much more closely related to NRIV than the prototype lab strain used in most studies to date. Based on analysis of our sequencing data we have made several interesting observation related to the emergence of NRIV, among which is that mutations in the non-coding regions of NRIV are not necessary to facilitate reassortment, something that would have presented a significant hurdle to future studies aimed at generating BUNV/BATV reassortant viruses using reverse genetics. Indeed, with the availability of these complete genome data we are proceeding with the construction of a full-length clone system for these viruses, which will in future allow us to construct various reassortant and chimeric viruses and thereby map the viral determinants associated with NRIV virulence and its ability to cause HF. Further, in order to identify measurable phenotypic characteristics that could be related to the acquisition of virulence by NRIV, we have previously analysed growth of BUNV and BATV in various relevant cell lines. In particular, we found that in C6/36 (mosquito cells) only a few BATV strains, including the UgMP-6830 strain, show efficient growth at early time points after infection. This could have implications for successful virus spread in the mosquito vector and potentially limit which BATV strains can generate NRIV-like viruses during reassortment. We are currently analysing the requirements for reassortment during co-infection in various cell lines, including both C6/36 cells and various mammalian cell types, using a co-infection/reassortment assay that we have established. These experiments will provide us with greater insight into where in the natural infection cycle reassortment occurs and what are the potential products of reassortment. This information will be used to guide our future studies of reassortment using the NRIV reverse genetics system as well as being used as a framework to understand the various other naturally occurring reassortant viruses that we have identified as part of our broad-scale phylogenetic analysis of the Orthobunyavirus genus, which is also currently ongoing. (2) Molecular determinants of host range and antigenic shift in Simbu serogroup viruses: The Simbu serogroup also belongs to the genus Orthobunyavirus and can be categorized into those viruses that infect i) humans (e.g. Oropouche virus), ii) livestock animals (e.g. Akabane virus) or iii) vertebrate hosts (rodents, birds, monkey etc.) other than humans or livestock (e.g. Mermet virus). In order to identify the viral determinants of host range among Simbu serogroup viruses, we have already determined full-length genome sequence of more than 20 Simbu-group viruses/strains, and bioinformatics analyses are ongoing. Oropouche virus (OROV) has caused more than 30 outbreaks with at least 500,000 cases identified between 1960 and 2009 in South America. It is currently unknown why OROV causes disease in humans while other Simbu-group viruses do not. Notably, during the 1999 Oropouche fever outbreak a novel Simbu-group virus, Iquitos virus (IQTV), was isolated from patients with Oropouche fever-like illness. This virus was a novel reassortant possessing L and S genome segments derived from OROV and an M genome segment of a novel Simbu group virus. Importantly, pre-existing neutralizing antibodies in people already infected with OROV do not neutralize and protect from IQTV infection. In order to elucidate the mechanism of antigenic shift associated with the emergence of novel genetic reassortants we have been carrying out genome sequencing on 39 OROV strains and 12 strains of IQTV. Our analyses have revealed that i) IQTV M segments are genetically close to those of OROV, compared with M segment sequences of other Simbu group viruses, but distant enough to belong to a distinct species, and ii) all OROV S genome segment sequences published to date (or registered in Genbank) are missing 200 nucleotides compared with the full-genome sequence that we completed using a combination of next generation sequencing and careful sanger-based sequencing. We are now developing a reverse genetics system for OROV based on our sequence results, which will represent a critical step towards characterization of the molecular determinants of host range and antigenic shift in Simbu serogroup viruses. (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 will conduct an extensive genetic analysis and biological characterization of uncharacterized taxonomically ungrouped bunyaviruses isolated from Africa, Asia, South and North America. During 2005-2013, Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) and Heartland virus, 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 Bhanja group viruses (BHAVs) and Uukuniemi group viruses (UUKVs), the epidemiological study and diagnosis of all TBPVs simultaneously has been difficult due to serological and genetic divergence among these viruses. Therefore, we expanded our extensive genome sequencing attempts to the various tick-borne phleboviruses and established a simple and fast detection method for multiple TBPVs by using one-step RT-PCR with a single pair of degenerate primers designed based on conserved virus genome sequences. Moreover, we identified novel TBPVs from uncharacterized bunyavirus samples and ticks corrected in Mali, strongly demonstrating that our simple RT-PCR system is of sufficient sensitivity and broad-reactivity to detect a range of TBPVs, including previously uncharacterized viruses that are divergent from known TBPVs. Our system will help field studies to identify both known and novel TBPVs, and can be used for the screening of uncharacterized febrile illnesses associated with tick bites.

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