Examining the Link Between Viral Genotype and Disease Phenotype Using Clinical Cases of HSV-1 and a Human Neuronal Model
Open Access
- Author:
- Shipley, Mackenzie Marie
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 24, 2018
- Committee Members:
- Moriah Louise Szpara, Dissertation Advisor/Co-Advisor
Moriah Louise Szpara, Committee Chair/Co-Chair
Melissa Rolls, Committee Member
Richard J. Frisque, Committee Member
John Warren Wills, Outside Member
Lorraine C Santy, Committee Member - Keywords:
- herpes simplex virus
genomics
infection
genital
neonatal
neurobiology - Abstract:
- Herpes simplex virus type 1 (HSV-1) is a lifelong neurotropic pathogen that affects millions of people all over the world, with a prevalence rate of up to 90% in some countries. This virus causes a chronic infection that can result in a variety of disease outcomes ranging from painful lesions on the skin, to infectious keratitis, and even deadly encephalitis. What is not yet understood is why such a wide spectrum of disease severity in those infected with HSV-1 is observed. It is likely that multiple factors such as the host immune system and viral genetics contribute to the clinical course of HSV-1 disease. As has been performed in human genetics with genome-wide association studies, the research presented in this dissertation sought to investigate a possible link between viral genotypes and disease outcomes (phenotypes) using clinical cases of HSV-1 and an in vitro human neuronal model. If the course of disease among individuals could be predicted based on particular genetic signatures in their HSV-1, this would result in improved diagnostic and therapeutic strategies using a personalized medicine approach. In work from this dissertation, a scalable human neuronal cell culture model was developed to investigate neuron-virus interactions using clinical isolates of HSV-1 in vitro. Unlike other available neuronal models, the differentiated SH-SY5Y model presented here is species-matched, chromosomally stable, homogeneous, and able to be scaled up or down for bulk biochemical or single-cell analyses. When this differentiated neuronal system was characterized and compared to the parental SK-N-SH, and undifferentiated SH-SY5Y progenitor cells, differences between neuronal protein expression and localization were identified, in addition to a disparity in HSV-1 replication and virus production. This neuronal model was also able to distinguish between virulent and less virulent strains of HSV-1 based on viral replication and cell-to-cell spread alone. These data suggested that this system might be advantageous for characterizing the relevant virulence of clinical isolates of HSV-1 in vitro, in addition to investigating specific neuron-virus interactions that may vary between strains. This dissertation also highlights research that utilized a targeted genomic enrichment strategy and next-generation sequencing (NGS) to explore viral genotypes and the amount of genetic diversity present in clinical samples of HSV-1. We used comparative genomics to analyze HSV-1 genomes derived from longitudinal viral shedding samples acquired from a woman who suffers from frequent and severe genital outbreaks. With this approach, we determined that HSV-1 genomes separated by multiple rounds of latency and reactivation, expanded in cell culture, or collected from different anatomical regions of the female genital tract were all highly identical to one another at the consensus genome level. In contrast, there was substantial variation in the amount of genetic diversity present in viral populations derived from the different sample types of genital HSV-1. This genetic diversity manifested as minority variants (MVs), or alternative alleles that exist below the consensus level at a particular nucleotide position. This viral genetic diversity appeared to be influenced by sample type—lesion, non-lesion, or cultured isolate—and host immune selection pressures or lack thereof. The same NGS and computational approach was used to analyze another clinical case of HSV-1, that of a perinatal transmission event between mother and neonate that resulted in dual fatality. From this study, we ascertained that the mother’s HSV-1 genome acquired late in her pregnancy and the virus that was transmitted to the neonate were nearly identical to one another at both the consensus genome and viral population levels. We also observed a small percentage of MVs that appeared to either be i) not transmitted from the mother to the neonate or ii) apparently acquired de novo in the neonate. Most of the MVs that we did track occurred at or near homopolymers or tandem repeats, suggesting a possible mechanism of HSV-1 evolution. While it is likely that the rare viremic infection in the mother and lack of established immunity in the neonate influenced the outcome of this perinatal transmission event, these data provide a foundation for future analyses of additional pairs of transmission samples. The findings from the studies included in this dissertation have shed light on how much genetic diversity is present in currently circulating HSV-1 and how this variation in the viral population may be linked to clinical disease outcomes and transmission events in people. To draw firm conclusions about the influence of viral genotype on the clinical course of disease (phenotype), more patient samples need to be analyzed using similar methodology and techniques as we present here. These findings will impact how future studies on HSV-1 are conducted, as we determined that expanding clinical HSV-1 samples in culture can lead to a bottleneck and subsequent drift in the viral population that is not representative of a natural infection. We also concluded that genomic analyses at the MV level as opposed to the consensus genome level provide more insight into the population dynamics and evolution of this large DNA virus. Ultimately, the data presented here generate new questions about the relationship between HSV-1 genotypes and clinical disease outcomes. Investigation of these questions will likely result in an improved understanding of the clinical course of HSV-1 disease and the potential production of new therapeutics and advanced diagnostic treatment strategies in the future.