Date of Graduation

5-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Food Science (PhD)

Degree Level

Graduate

Department

Food Science

Advisor/Mentor

Gibson, Kristen

Committee Member

Ramena, Grace

Second Committee Member

Forbes, Kristian

Third Committee Member

Kwon, Young

Keywords

Human Norovirus; Lactobacillus salivarius; Replication; Tulane Virus; Zebrafish Embryo; Zebrafish Larvae

Abstract

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis worldwide; however, the lack of robust laboratory culture systems and reliable animal models has limited progress in understanding viral replication and developing effective intervention strategies. While alternative models such as the zebrafish system have been explored, gaps remain in evaluating their ability to support replication across diverse HuNoV strains and in identifying factors that influence infectivity. This dissertation provides a comprehensive investigation into the use of the zebrafish model for studying viral replication, host interactions, and potential inhibition strategies. Initial studies focused on optimizing viral delivery methods using Tulane virus (TuV) as a surrogate due to the limited availability of HuNoV positive stool specimens. Three delivery approaches (microinjection, immersion, feeding) were evaluated, demonstrating that microinjection was the only method that supported measurable viral replication. Microinjection of 3 nL of TuV into zebrafish embryos (< 6 hours post-fertilization) resulted in significant replication, with viral RNA levels reaching 6.22 log RNA copies per embryo at 4 days post-infection. Further analysis revealed that TuV replication was dependent on developmental stage, with replication observed in zebrafish embryos but not in larvae (3 days post-fertilization). This difference may be linked to developmental changes in sialic acid expression, as TuV binds α-2,6-linked sialic acids expressed in embryos but not in larvae. However, blocking these receptors with Sambucus nigra lectin did not reduce replication, suggesting alternative entry mechanisms. Building on these findings, the replication of twenty-six HuNoV positive stool specimens representing 5 GI and 7 GII strains were evaluated in zebrafish larvae. The model supported replication of a majority of strains, including 3 GI, 2 GII.4 and 3 non-GII.4 genotypes, although replication was not observed for all stool specimens within the same genotype. Zebrafish embryos were not suitable for these studies due to mortality following stool microinjection, likely resulting from stool associated toxicity. The variability in replication among strains and stool specimens indicates that factors beyond viral genotype, such as stool composition, influence replication outcomes in this system. The dissertation further examines the potential for gut microbial inhibition of HuNoV replication using Lactobacillus salivarius. Treatments containing bacterial cells and overnight broth significantly reduced viral RNA levels (P < 0.01), whereas cell free supernatant showed no inhibitory effect. An in vitro binding assay demonstrated that 67% of the virus physically associated with L. salivarius. Additionally, both live and heat-killed bacteria exhibited similar inhibitory effects, suggesting that bacterial viability is not required for inhibition. Overall, this work highlights the utility of the zebrafish model as a scalable and versatile platform for studying HuNoV replication across diverse strains and for evaluating antiviral strategies. The findings emphasize the influence of delivery method, developmental stage, and specimen-specific factors on viral replication and provide important insights that support the continued development of model systems for advancing HuNoV research.

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