Thermal Selection of Seneca Valley Virus Gives Rise to a Novel Thermostable Mutant
This project aimed to improve the stability of the novel oncolytic virus, Seneca Valley virus (SVV). This was with the overarching goal of determining the residues and mutations thereof within the viral capsid that are responsible for capsid stability, to inform the future production of SVV Virus-Like Particles (VLPs) for use as a drug delivery vector. The thermal stability of wild-type SVV-001 was first investigated to determine a baseline to which thermostable mutants could be compared, and to inform a regimen of heating and passage to derive thermostable mutants. During optimisation of these initial experiments, heating of a sample containing approximately 107 PFU/mL SVV-001 to 58.5 °C for 30 minutes produced a single viral plaque. Virus collected from this plaque was shown to be resistant to heating at 56 °C, an improvement on the wild-type, which was shown to lose approximately 99% viral titre to heating at 53.5 °C for 30 minutes. This thermostable phenotype was also confirmed using Particle Stability Thermal Release Assays (PaSTRy). Attempts to select for increasingly thermostable virus were unsuccessful. Mutant virus was purified and the capsid-coding region of the genome sequenced. This revealed four mutations in the thermostable mutants. One of these mutations, A1776G, was predicted to have an effect on the maturation of capsid proteins, which was supported by initial results of SDS-PAGE gel analysis. The other three mutations were either synonymous mutations well-conserved in Senecavirus isolates, or outside of the capsid coding region of the genome, and so were of limited applicability to the production of VLPs.Thermostable virus was then optimised for cryo-electron microscopy to determine its structure, with a low-resolution structure derived as a proof-of-principle.Future studies are warranted to determine if the A1776G confers a thermostable phenotype to the wild-type virus, and in turn to the SVV VLP. The basis for this phenotype should also be investigated, as well as a means to introduce the desired drugs into the genomeless capsid.
Advisor: Bostina, Mihnea
Degree Name: Master of Science
Degree Discipline: Department of Microbiology
Publisher: University of Otago
Research Type: Thesis