Year of Award

2025

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Brent Ryckman

Commitee Members

J. Stephen Lodmell, Jay Evans, Scott Wetzel, Jeffrey Good

Keywords

Coinfection, Human cytomegalovirus, Recombination

Abstract

Human cytomegalovirus (HCMV) is the largest of any human virus, both physically at up to 300 nm in diameter, and as a genome, with its 235 kbp DNA genome with at least 165 open reading frames. It is also the number 1 non-genetic cause of birth defects and has a global prevalence rate of just over 80%. Despite these characteristics, it is not as well known to the general public as some of the other human herpesviruses, such as herpes simplex or varicella-zoster virus. Like other herpesviruses, HCMV can establish latency, leading to a lifelong infection for its host with occasional reactivation. An infection also does not protect against future exposures, so a host may be infected multiple times throughout their life. These infections can be from different strains, as HCMV is a highly diverse virus, and infections with multiple strains have been shown to correlate with worse health outcomes for patients. However, studying infections with multiple strains in a laboratory setting has not been well studied. The research in this dissertation will help to build a foundation on which future work exploring the effect of diversity on infection can be built. It begins by exploring methods to detect coinfections, where a host cell is infected by more than one virus. Chapter 2 introduces single color assays and trans-complementation, both situations in which coinfection must be inferred from the data, and infections with multiple virus stocks expressing different fluorescent proteins, where coinfections can be counted directly by flow cytometry. The third chapter explores the use of single color assays to quantify the apparent cooperation between virions and offers three possible hypotheses for what the driving force is for cooperation. These are tested, in silico, by simulations created by our collaborators, and results suggest that 2 of the 3 hypotheses warrant further exploration at the bench. The final chapter draws on the recombinogenic nature of HCMV genome replication, its propensity for coinfection, and fluorescence-activated cell sorting to create a library of recombinant progeny derived from the parental strains TB40/E-BAC 4 and ME. Course genotyping confirms that recombinants were isolated from the progeny stock and, coupled with several phenotype assays, the viruses will assist in determining which genes are responsible for phenotypes such as spread mode and cell-free infectivity. These characteristics are typically correlated with the relative expression level of two receptor-binding complexes, and one recombinant shown breaks the correlation. The techniques described are applicable to other virus systems and will allow others to examine cooperativity between virions in their own virus species.

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