Date of Award

12-1-2022

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (vii, 49 pages) : illustrations (chiefly color)

Dissertation/Thesis Chair

Andrew Berglund

Committee Members

Melinda Larsen, David VanHoute, Jared Richardson

Keywords

Activity, Bioinformatics, Nanopore, VirCy-Seq, Virus, Pharmaceutical microbiology, Pharmaceutical technology, Viruses, Nanopores

Subject Categories

Biology | Molecular Biology | Virology

Abstract

The main purpose of pharmaceutical production is to produce safe effective medicine for patient use. In an effort to ensure patient safety constant surveillance for viruses takes place. The detection of a viral nucleic acid in a pharmaceutical production setting results in investigations to assess its infectious potential. This is an intensive, expensive process that entails many tests such as the observation of hemadsorption, cytopathic effects (CPE), and more. These tests are typically specific and only capture certain viruses, as factors like CPE can only be observed in some viral species. A new investigational method that is effective on all viruses is needed. Viruses vary greatly from one another, with how they: affect organisms, replicate, etc.. A universal feature of viruses is that they have genomes and capsids which need to be reproduced to replicate. These production of viral genomes and capsids relies on nucleic acid intermediates. While the intermediates differ from their genomes in either strand or nucleic acid type, they can be used to determine viral activity. As an example, if RNA was detected with Group I viruses of the Baltimore Classification system, it would show their dsDNA genome is being transcribed and thus active. A protocol that enables the identification of nucleic acid types would act as an investigational tool for all virus species. VirCy-Seq is a protocol that aims to do this and replace the short scope assays currently in use. VirCy-Seq utilizes a previous protocol, Simul-Seq, process of distinctly labeling nucleic acids, with changes to the protocol that adapt it to the Nanopore platform and take advantage of its ability to sequence long reads. VirCy-Seq also has a custom bioinformatic pipeline that identifies nucleic acid types for each virus so the replicative intermediates of viruses could be easily identified. Research has optimized the VirCy-Seq protocol to be accurate and effective at identifying nucleic acid types. VirCy-Seq’s accuracy was assessed using representative DNA and RNA sequences that allowed verification of findings. Issues with VirCy-Seq such as mislabeling of DNA and low identification of DNA reads were resolved through experiments that compared factors surrounding the issue. VirCy-Seq was able to detect PI2 infection in samples that while infected with PI2, would be labelled non-infectious by cell culturing methods. VirCy-Seq was able to identify distinct differences between two sample groups that could imply differences in their viral activity. These findings support VirCy-Seq as an investigational tool for characterizing viral activity in all viral infections.

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