Date of Award:

5-1-1993

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology (Molecular Biology)

Committee Chair(s)

Joseph K.-K. Li

Committee

Joseph K.-K. Li

Committee

Thomas D. Bunch

Committee

Nabil N. Youssef

Committee

Dennis L. Welker

Committee

Vijendra K. Singh

Abstract

Despite antigenically distinct serotype-specific neutralization, sequence analyses indicated that four of the five United States bluetongue viruses (serotypes 10, 11, 13, and 17) originated from a single, common gene pool. Bluetongue virus 2 belongs to a second distinct gene pool. These genetic distinctions are reflections of the geographic distribution of bluetongue viruses in North America. This study also focused on the neutralization epitopes of serotype 13, the strain endemic in the western United States. Oligoclonal antibodies against MAP-constructed synthetic peptides and polyclonal antibodies against denatured viral proteins failed to exhibit neutralizing activities. However, monoclonal and polyclonal antibodies against inactivated purified virions exhibited neutralizing activity to the homologous virus but not to the heterologous viruses. These results suggest that the neutralization epitopes are serotype specific and conformation-dependent. Multiple antigenic epitopes on the major cognate outer capsid proteins, VP2s, of bluetongue viruses were mapped and identified by employing monospecific site-directed antibodies and competition with sequence-specific synthetic peptides. Several internal antigenic domains were identified and may be involved in inter- and/or intramolecular interactions. Competition with sequence-specific synthetic peptides identified only one linear synthetic peptide (EMDDDETEYE), corresponding to amino acids #642-651 of VP2 of bluetongue virus 13 that blocked both the neutralizing activity of monoclonal antibody D24.15 and its ability to bind to VP2 of serotype 13. This epitope was also located on the surface of serotype 13 purified virions. An interpretation of these data is that the major neutralization determinant of the outer capsid protein on the virion is a conformation-dependent and serotype-specific epitope. It was also determined that several hydrophilic regions present on VP2, including amino acids #397-408 and #428-442 of VP2 of serotype 2; #199-213 and #248-265 of VP2 of serotype 10; #82-94 and #310-321 of VP2 of serotype 11; and #31-43 and #444-464 of VP2 of serotype 17, were surface accessible and potential neutralization epitopes of these four bluetongue viruses.

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