Author ORCID Identifier

https://orcid.org/0000-0001-5050-0183

Year of Publication

2019

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Veterinary Science

First Advisor

Dr. Thomas M. Chambers

Abstract

Equine herpesvirus-1 (EHV-1) is one of the most important and prevalent viral pathogens of horses causing a major threat to the equine industry throughout most of the world. EHV-1 primarily causes respiratory disease but viral spread to distant organs enables the development of more severe sequelae; abortion and neurologic disease. In order to produce disease, EHV-1 has to overcome the innate barrier of the type-I interferon (IFN) system in host cells. However, the underlying mechanisms employed by EHV-1 to circumvent the type-I IFN response in host cells are not well understood. In this project study, using molecular techniques, we explored how EHV-1 is able to escape the type-I IFN response in host cells during infection. We also investigated whether EHV-4, a closely related but less pathogenic virus, has similar effects on type-I IFN as a clue to understanding how widespread IFN suppressive function is found among equine alphaherpesviruses.

Our data showed that inhibition of the type-I IFN response in host cells is not a function of neuropathogenicity of EHV-1 strains. However, a reduced type-I IFN response correlated with pathogenicity as EHV-4, unlike EHV-1, was unable to down-regulate the type-I IFN response in equine endothelial cells (EECs). Investigation of the mechanisms employed by EHV-1 to suppress type-I IFN revealed that the virus sequentially prevented outside-in signaling events that lead to type-I IFN production. Specifically, EHV-1 blocked the expression of Toll-like receptors (TLR) 3 and TLR4 at 6 hours post-infection (hpi) and 12 hpi. EHV-1 also prevented the transcription of IRF7 and IRF9 at different time-points during infection. The virus also perturbed the JAK-STAT signaling pathway by negatively regulating the cellular levels of TYK2 and phosphorylation-mediated activation of STAT2 molecules. Immunofluorescence data revealed that during infection, EHV-1 was able to sequester STAT2 molecules from nuclear translocation. This may be a limiting step preventing the formation of interferon- stimulated gene factor 3 (ISGF3) whose nuclear translocation is required to transactivate interferon-stimulated genes (ISGs) including IRF7.

Further investigation showed that unlike EHV-1, EHV-4 only interfered with phosphorylation-mediated activated STAT1 and STAT2 molecules at 3 and 6 hpi. EHV-4 was unable to block TLR3/4 and IRF7/9 mRNA expression at any time-point. Intriguingly, while viral late gene of EHV-1 mediates inhibition of STAT phosphorylation, our data showed that for EHV-4, a virus late gene did not mediate the inhibition of STAT phosphorylation. The findings from this study help illuminate how EHV-1 strategically interferes with limiting steps required for type-I IFN response in host cells to promote pathology. Our data also strengthen the hypothesis that the ability to shut off host factors required for type-I IFN production might be directly related to the degree of pathogenicity of the EHV subtypes.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2019.374

Funding Information

This work was supported by the USDA National Institute of Food and Agriculture, Hatch project no. 1011380. It is a project of the Kentucky Agricultural Experiment Station (project no. KY014053), supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Animal Health Program. I was also supported by grant from the American Quarter Horse Association Young Investigator Award and by a fellowship from the Geoffrey C. Hughes Foundation.

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Virology Commons

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