Ecology of Hantaviruses in Rodent Reservoirs and Their Early Innate Immune Responses in Human Model Systems

The spillover of zoonotic RNA viruses is responsible for a great deal of the disease outbreaks in human populations. These spillover events are set to continue due to anthropogenic and environmental changes that impact the distribution of these viruses. The viruses in the family Hantaviridae are classified as one of these emerging zoonotic RNA viruses. The spillover of the viruses in this family are responsible for two severe human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These viruses are distributed across the globe and are responsible for a large number of human disease cases with potentially high mortality rates each year. Unfortunately, there is a lack of surveillance efforts to identify hantaviruses in most countries making accurate diagnosis or recognition of hantavirus cases complicated. To address the potential public health impact of hantaviruses, we surveyed rodents in a rural region of Ukraine, and identified a high prevalence which underscores the potential for human disease in this country. As it is challenging to address how hantaviruses infect humans, I established approaches to evaluate the early innate immune response in primary lung microvascular endothelial cells (HLMVECs) with pathogenic and nonpathogenic hantaviruses. Surprisingly, my findings challenged some of the current dogma in that there were not dramatic difference between pathogenic and nonpathogenic viruses. This work highlights the critical need for advancement of cell culture models to probe the immune response. To understand the ecology of hantaviruses in their reservoirs their prevalence was studied in northwestern Ukraine. A field capture study was conducted at two sites which each had distinct habitats and contained nine capture lines. During this survey, we captured 424 small mammals, consisting of species across three orders. The most abundant species were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%). Out of the collection, it was determined that 79 animals were seropositive by immunofluorescent assay (IFA), from which 15.7% were M. glareolus, 20.5% A. flavicollis, and 33.9% A. agrarius. These finding were of interest as M. glareolus and Apodemus spp. harbor Puumala orthohantavirus and Dobrava-Belgrade orthohantavirus viruses, respectively, which are responsible for causing HFRS in humans. IFA reciprocal titer showed a wide distribution indicating new infections are occurring. No relationship was found between species diversity and the proportion of hantavirus seropositive animals captured at these sites. Population analysis on M. glareolus and Apodemus spp. revealed that neither sex nor age was associated with being seropositive. To define the early innate immune responses during human infection by hantaviruses, this research studies the responses in HLMVECs, the primary cells of infection in humans, infected by the pathogenic viruses, Andes orthohantavirus (ANDV) and Hantaan orthohantavirus (HTNV), and the nonpathogenic virus, Prospect Hill orthohantavirus (PHV). A curated list of 39 host genes were studied across multiple time points during the first 72 hours of infection of HLMVECs from a male donor by these three viruses. mRNA level analysis revealed the mRNA levels of only CCL5, CXCL10, CXCL11, IDO1, IFNB1, IRF7, and TLR3 we increased during infection of each viruses. The measurement of CCL5, CXCL10, CXCL11, IDO, and IFN-β secreted protein levels in the same HLMVEC donor during infection confirmed gene expression findings. The study of host immune responses to hantavirus infection was expanded to include HLMVECs from an additional male and two female donors. Measurement of secreted protein levels of CCL5, CXCL10, CXCL11, IDO, and IFN-β by each of the four donors revealed that levels of these proteins are upregulated during infection by each of the viruses. Pair wise analysis on these secreted protein levels by each of the donors during hantavirus infection suggests that donor characteristics and virus species together drive different outcomes. However, female donors had higher levels of CXCL10, IDO, and IFN-β and these increased protein levels were species specific. Lastly, the suppression of immune response involved in cell death were examined and it was found that ANDV is capable of inhibiting cell death in HLMVECs. In summary, the findings presented, show the critical need to understand and define the early innate immune responses to hantaviral infection in human models as well as the necessity of understanding the ecology of hantaviruses in their reservoir hosts.