Date of Award:

5-2026

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Veterinary Clinical and Life Science Department

Committee Chair(s)

E. Bart Tarbet

Committee

E. Bart Tarbet

Committee

Brett L. Hurst

Committee

Stephanie Martinez

Abstract

Influenza virus infections can be treated with antiviral medications, and researchers are exploring whether combining two antiviral drugs can improve treatment effectiveness. Sometimes, drug combinations work better than the sum of their individual effects (called synergy). However, other times the drugs can interfere with each other, making the treatment less effective (called antagonism). In this research, oseltamivir was combined with either peramivir or baloxavir as treatments for mice infected with the 2009 H1N1pdm influenza virus. These drugs have different mechanisms of action: oseltamivir and peramivir prevent the virus from leaving infected cells, while baloxavir inhibits the virus from replicating its genome.

Because treatment effectiveness in animal studies is often measured using “survival,” it is important to understand how survival is defined. In mouse studies, a common humane endpoint is a predefined weight-loss cutoff, meaning animals are euthanized once they lose a certain percentage of their body weight. This cutoff can influence how survival outcomes are interpreted, since different thresholds may change whether a mouse is considered to have survived. Therefore, the effects of different weight-loss cutoffs were evaluated in studies examining oseltamivir combined with peramivir and oseltamivir combined with baloxavir marboxil.

Each drug was administered as monotherapy (alone) and in combination for five days post-infection in BALB/c mice. When given as monotherapy, high doses of all three drugs vi resulted in 100% survival, while low doses caused survival rates ranging from 0-60%. In the peramivir—oseltamivir study, when 1, 0.3, and 0.1 mg/kg/day doses of peramivir were combined with 0.3 mg/kg/day of oseltamivir, the combinations performed better than either drug alone. However, at higher doses, the combination was not always more effective than monotherapy. In the baloxavir—oseltamivir experiment, results were mixed. Although both drugs worked well individually, their combination did not consistently improve survival. The most notable synergy was observed when oseltamivir at 0.3 mg/kg/day was combined with baloxavir at 0.3 mg/kg/day, resulting in complete protection.

The weight-loss cutoff used to determine “mortality” significantly influenced how the effectiveness of drug therapies was interpreted. Influenza-infected mice lose weight as the infection progresses, so severe illness is gauged by the percentage of weight lost, such as 20%, 25%, or 30%. When a specific weight loss threshold is reached, infected mice are euthanized to minimize their suffering. However, adjusting these cutoff points affected the outcomes of drug combination therapy. In some cases, a drug combination seemed synergistic with one weight-loss cutoff but antagonistic with another. MacSynergy and SynergyFinder, software programs used for visualizing and interpreting drug synergy, confirmed that the percentage of weight loss used to define mortality could make the same treatment appear synergistic, neutral, or antagonistic. This underscores the importance of using standardized methods when testing antiviral combinations in animal studies.

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