Document Type

Thesis - Open Access

Award Date

2017

Degree Name

Master of Science (MS)

Department / School

Veterinary and Biomedical Sciences

First Advisor

Alan Young

Keywords

EHDV, monoclonical antibodies

Abstract

The purpose of this project was to define monoclonal antibodies against viral proteins from Epizootic Hemorrhagic Disease Virus (EHDV) to be used in quality control of vaccines produced by an industrial partner, and development of tools to identify EHDV. EHDV is an Orbivirus, which also includes Bluetongue Virus and African Horse Sickness, which are all transmitted through arthropod vectors. EHDV causes significant morbidity and mortality in white-tailed deer, but has recently been found to infect cattle. EHDV recently caused significant outbreaks affecting both the farmed and wild cervid industry, however few reagents and tools exist to protect against this disease or define the virus in vitro. To develop new, efficient reagents that can be used to identify EHDV, we identified and tested monoclonal antibodies against two different viral capsid proteins, VP2 and VP5. To do this, predictive algorithms were used to generate immunogenic peptides specific for each protein. Three groups of mice were used, one injected with the VP2 peptides, one with the VP5 peptides, and the third with a proprietary marker sequence localized to the amino terminus of recombinant proteins produced by the industrial partner. After fusion, 192 wells were plated from each fusion. Wells containing growing cells after about sixteen days were screened and positive wells were subcloned. This process was repeated twice, and we were left with two hybridoma clones recognizing epitopes of VP5 and one of VP2. Further testing showed cross reactivity the two VP5 antibodies, so we focused on the monoclonal antibodies directed against VP2, line 13-2. Following generation of ascites, utility in recognition of both our partners’ recombinant VP2 protein and native VP2 proteins of the virus was confirmed. In addition, a commercial VP5 antibody directed against African Horse Sickness virus also recognized recombinant protein, and native EHDV VP5 protein in multiple assays. Both antibodies reacted by ELISA against the native proteins and viral lysate from both EHDV-1 and EHDV-2, although only the VP5 antibody showed the ability to recognize viral proteins by Western Blot. Work is underway to define the ability of the antibodies to recognize and identify EHDV by IFA.

Library of Congress Subject Headings

Ruminants -- Virus diseases.
Communicable diseases in animals.
Monoclonal antibodies.
Monoclonal antibodies -- Diagnostic use.

Description

Includes bibliographical references (pages 37-38)

Format

application/pdf

Number of Pages

46

Publisher

South Dakota State University

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Rights Statement

In Copyright