Date of Award:

5-1-1992

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 A. Grover

Committee

Gregory J. Podgorski

Committee

Vijendra K. Singh

Committee

Jon Y. Takemoto

Abstract

The identification of antigenic epitopes on VP7 and VP5, serogroup and serotype-specific antigens of bluetongue virus (BTV), respectively, was studied using the combination of polyclonal, monoclonal and oligoclonal antibodies and competitive blocking studies. This research is the first to identify the antigenic epitopes on proteins of any orbivirus. Two distinct antigenic determinants have been identified on VP7, the major inner capsid protein. One of the antigenic epitopes, consisting of eleven amino acid residues, was located between residues 339 and 349 at the carboxyl terminus of VP7. The location of this epitope was confirmed by specific inhibition of polyclonal antibody binding to VP7 using a synthetic peptide (LTRAIARAAYV). The second epitope of VP7, identified by a sequence-specific oligoclonal antibody (R-AG-18) and a monoclonal antibody (45.4.1), was mapped between residues 122 and 139. These two epitopes were highly conserved on the VP7 protein among all five U.S. BTV serotypes. One linear (continuous) antigenic epitope, located between residues 175 and 189 (ALQREAAERSEDEIK) of the VP5 protein, was conserved in all five U.S. bluetongue viruses. The oligoclonal antibody (AK-15) raised against this linear epitope reacted with the denatured VP5 protein on Western blots as well as native one on the surface of purified BTV virions. A second epitope, present in BTV-2, -10, -11, and -17, but not in BTV-13, was identified by a monoclonal antibody (34.7). The binding of the monoclonal antibody to VP5 could be specifically blocked by three short synthetic peptides corresponding to residues 33 to 42 (KAAERFAESE), 159 to 168 (EKILKEEDSK), and 206 to 215 (EIERDGMQEE), indicating that this antigenic epitope identified by the monoclonal antibody (34.7) is conformational (discontinuous) However, the oligoclonal antibody (AK-15) and the monoclonal antibody (34.7) did not neutralize BTV viral infectivity as determined by a plaque reduction assay.

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