Document Type

Dissertation - University Access Only

Award Date

2004

Degree Name

Doctor of Philosophy (PhD)

Department / School

Veterinary and Biomedical Sciences

First Advisor

Christopher C. L. Chase

Abstract

Bovine herpes virus type 1 (BHV-1), a member of the alphaherpesvirus group, is an economically important pathogen of cattle and is responsible for a variety of clinical symptoms including respiratory disease, reproductive tract lesions and abortion. Several hypotheses have been advanced to explain the cytopathology of BHV-1, however the mechanisms of BHV -1 cytopathogenicity remains unknown. The aim of this work was to investigate the mechanism involved in BHV-1 pathogenesis and to understand the mechanism for the viral infection. Our study focused on two envelope viral glycoproteins, gE and gI, and how these glycoproteins interact with the cellular cytoskeleton, their level of phosphorylation and expression of these glycoproteins during BHV-1 infection.

The first objective was to investigate the effect of wild-type (WT) or mutant strains (Vk36 [gI], Vk25 [gE], gEΔ3.1 [gE], & v20 [gI and Ge]) of BHV-1 on the integrity of cytoskeleton components, actin filaments (AF), microtubules (MT) and intermediate filaments (IF) during the course of viral infection. The results showed that the architecture of AF and MT were changed during the course of viral infection. It was also found that the glycoproteins gE and gI have a role in these changes. These glycoproteins triggered changes in the organization of cytoskeleton elements when they were transfected into Madin Darby bovine kidney (MDBK) cells. The gE and gI were associated with the soluble and. insoluble form of cellular actin. Our data demonstrated that WT or mutant strains of BHV -1 did not alter the quantity of cellular actin. None of the BHV-1 strains had an effect on the organization of IF or integrity of the Golgi apparatus.

Cytoskeleton inhibitors were used to investigate their effects on the architecture of cytoskeleton in BHV-1 infected cells. The results indicate that the cytochalasin D (CD), an actin inhibitor, had no effect on the expression of gE or gI. Destroyed actin filaments (AF) in CD-treated-infected cells abolished the effect of WT or mutant strains \ of BHV-1 on the actin filaments of MDBK cells. CD also disrupted the cell junction region between cells suggesting that the AF play a role in cell-to-cell adhesion. This disruption impaired the localization of gE in Vk36 (gI-)-infected cells. CD had no effect on the virus assembly with WT or mutant strains of BHV-1.

The depolymerization and stabilization caused by nocodazole (NOC) or taxol (TAX) affected the architecture of microtubules (MT) in mock and BHV-1 infected cells. •The effect of these inhibitors was dominant over the changes induced by BHV-1 infection. NOC and TAX also lowered the titers of cell-associated -virus and intracellular virus indicating that the MT could have a role in the processes of assembly and release in WT and mutant strains of BHV -1. Differences in the ratios of extracellular to cell associated virus among BHV-1 strains indicated that gI and gE had no role in the processes of assembly and egress.

The second objective was to investigate the role of phosphorylation in BHV-1 infection. Our data showed that among the protein tyrosine kinase inhibitors, genistein (GEN) and AG 17 reduced the replication of all BHV-1 strains. The WT strain was inhibited the most by GEN while gEΔ3.1 (gE ) was inhibited the least. Staurosporine (STP), a serine/threonine inhibitor, reduced the virus production in all strains. In the presence of GEN or STP, the phosphorylation of tyrosine or serine residues in gE and gI was markedly suppressed. Also, the expression of early viral protein gD or late proteins gI, gE, or gC was reduced in the presence of STP or GEN. Finally, the phosphorylation of tyrosine or serine residues in viral proteins (gE or gI) may be essential for WT-BHV -1 spread from cell-to-cell.

The results obtained in this study increased our knowledge about understanding the mechanism of BHV-1 pathogenesis. This new information will help in gaining control over BHV-1 infection.

Library of Congress Subject Headings

Cattle -- Virus diseases.
Herpesvirus diseases in animals.
Glycoproteins.
Cytoskeleton.
Phosphorylation.

Publisher

South Dakota State University

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Rights Statement

In Copyright