Date of Award

4-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Colleen B. Jonsson, PhD

Committee

James S. Adelman, PhD; Elizabeth A. Fitzpatrick, PhD; Richard J. Webby, PhD; Michael A. Whitt, PhD

Keywords

genetic diversity, habitat degradation, hantavirus prevalence; Interior Atlantic Forest, next generation sequencing, species diversity

Abstract

Understanding how climate and human land use change impacts trophic interactions is essential in the face of ongoing biodiversity loss and the continued emergence of RNA viruses and zoonosis. Human land use changes such as agricultural expansion, deforestation, and habitat degradation all have been linked to zoonotic disease emergence. Despite these commonalities, our mechanistic understanding of what drives them is lacking. Moreover, genomic surveillance in wildlife populations is critical for risk assessment and mitigation. Herein, using data collected from field studies within the Reserva Natural del Bosque Mbaracayú (RNBM), a protected area of the Interior Atlantic Forest (IAF), I examined the impact of predator exclusion and habitat degradation on the rodent community and the prevalence of hantaviruses within two reservoir hosts: Akodon montensis and Oligoryzomys nigripes. These species harbor Jabora hantavirus (JABV) and Juquitiba hantavirus (JUQV) respectively. Secondly, I developed an amplicon-based, next generation sequencing approach to survey the full-length genomes of these viruses in wildlife. In the first study, predator exclusion was conducted at three grid pairs with different levels of habitat degradation: least, moderately, and most degraded. Three grids were selected for experimentation and the others as controls. On experimental grids, electric fencing and cage traps were set up at each grid. This was done to exclude the entry/re-entry of terrestrial mammals. From these studies we found that predator exclusion did not impact the structure of the rodent community, nor did it alter hantavirus prevalence. Instead, habitat degradation altered hantavirus prevalence in Akodon montensis populations. We did not observe the same effect for Oligoryzomys nigripes. Additionally, we found that prevalence is strongly associated with host biological factors including sex and age. For both species, adult males were more likely to be infected. Using samples collected from these field studies, I used empirical approaches to determine whether a multiplexed-PCR based amplicon sequencing approach could be leveraged to sequence hantaviruses from which no sequence information exists. To do so, I manually designed degenerate primers to amplify JABV and JUQV. This allowed for the recovery of near complete full-length genomes for these segmented viruses. To demonstrate the utility of this tool, I screened 98 lung O. nigripes lung tissues for the presence of S and M segment vRNA. In total, I identified 16 RNA positives, eight of which were previously confirmed as being antibody positive. From these samples, I recovered full-length S and M segment genomes at an average depth of coverage >500x which facilitated the examination of the minority variant population. From all sequences collected, there were 33 synonymous, two nonsynonymous (V179I, I289T), and 20 3’ NCR mutations in the S segment sequences. There were 143 total mutations in the M segment. Of these, 137 were synonymous and six were nonsynonymous mutations: T104A, S310P, L340V, A370T, V504I, and S511L. Interestingly, nonsynonymous M segment mutations were identified in the glycoprotein, Gn, which is the spike protein involved in cellular attachment and entry. There was evidence of strong purifying selection acting on both genomic segments. There was no evidence of positive selection. Despite there being evidence of strong purifying selection, genetic diversity differed significantly by habitat type. Diversity was higher in the least degraded forests compared with the moderately or most degraded forests. Based on previous studies, there was no evidence to suggest that this level of diversity was caused by an increase in virus prevalence or host abundance. Instead, field data suggests that difference in host behavior may be mediating these responses. Altogether, this data suggests that there are tight constraints placed on the evolution of hantaviruses within their host. Long term, the sequencing approach described herein could be used for the surveillance of other segmented RNA viruses within their reservoir or non-reservoir hosts in nature. Moreover, examination of virus diversity at the population level can help elucidate mechanisms underlying virus persistence.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-6258-0093

DOI

10.21007/etd.cghs.2023.0623

Additional Files
2023-010-Spruill-Harrell-DOA.pdf (242 kB)
Declaration of Authorship
Chapter 3 Supplemental Predator Database.xlsx (99 kB)
Chapter 3 Supplemental Predator Database
Chapter 3 Supplemental Tables.Viruses.pdf (116 kB)
Chapter 3 Supplemental Tables.Viruses
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