Date of Award

January 2026

Document Type

Open Access Thesis

Degree Name

Master of Public Health (MPH)

Department

School of Public Health

First Advisor

Chantal B. Vogels

Second Advisor

Nathan Grubaugh

Abstract

Dengue virus (DENV) is a mosquito-borne RNA virus and increasing public health threat. Due to its error prone replication, single-nucleotide variants (iSNVs) are rapidly generated within the vector, leading to heterogeneity in viral populations, fitness, virulence, and transmissibility, including under vector control interventions. One such control strategy is the large-scale deployment of Wolbachia-infected mosquitoes worldwide. Wolbachia is an endosymbiotic bacterial biocontrol method that provides incomplete inhibition of DENV replication and transmission. Given its incomplete inhibition, it is critical to understand how Wolbachia impacts DENV-2 intra-host viral diversity. Additionally, successive blood meal feeding may influence intra-host viral diversity by supporting increased viral dissemination from the midgut. Here, I tested whether Wolbachia infection (wAlbB) and feeding behavior independently or interactively shape DENV-2 genetic variation. I conducted next-generation metagenomic sequencing of DENV-2 populations from bodies and legs of wildtype and wAlbB Ae. aegypti mosquitoes following a single infectious blood meal or a second non-infectious blood meal. High titer samples were identified by RT-qPCR and normalized for viral genome equivalents to reduce depth-driven bias and analyzed with iVar to call iSNVs (≥3% frequency and ≥400x depth). Diversity was quantified using iSNV and amino acid genetic distances, Shannon entropy (Sn), and dN/dS ratios. Selected nonsynonymous iSNVs present in the global DENV-2 Nextstrain dataset were evaluated using AlphaFold Server structural modeling. Overall, neither Wolbachia nor feeding behavior had a great impact on DENV-2 intra-host diversity. However, wAlbB single-fed leg samples demonstrated a statistically higher iSNV frequency, indicating possible Wolbachia-induced selection. Additionally, structural predictions suggested that envelope substitution N124Y could potentially affect envelope dimerization and thus, possibly transmission. This work provides insights into how feeding behavior and Wolbachia shape DENV-2 viral evolution within the mosquito, informing arbovirus evolution and intervention strategies.

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This is an Open Access Thesis.

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