Molecular and immunobiological characterization of VP8*, n-terminal trypsin cleavage product of bovine rotavirus VP4
Date
1996-01-01
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ORCID
Type
Degree Level
Doctoral
Abstract
It is the objective of this thesis to contribute to the understanding of the molecular pathogenesis of rotavirus infection and the development of a subunit vaccine for the control of rotavirus infection. Rotavirus, a member of the family Reoviridae, is a major etiological agent of diarrhea in animals. It has been shown that VP8*, the N-terminal trypsin cleavage product of structural protein VP4 is responsible for virus binding to MA-104 cells and human type O erythrocytes. The VP8* gene was cloned and expressed in bacteria. This protein (rVP8*) was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). To study the interaction between rVPB* and cells, binding and inhibition assays were performed. The rVP8* bound to MA-104 cells and the viral protein binding was competitively inhibited by BRV. Permissive cell binding was not disrupted by neuraminidase treatment of the MA-104 cell monolayers, which suggested that the binding of BRV to MA-104 cell is sialic acid independent. To study the interactionbetween VP8* and erythrocytes, rVP8* was solubilized and hemagglutination (HA) and hemagglutination inhibition (HI) assays were performed. The rVP8* protein agglutinated erythrocytes and its HA was inhibited by antiserum to BRV. Molecular and biochemical characterization of the HA demonstrated that rVP8* formed dimers, and its binding to erythrocytes required an α2-8 linkage between sialic acids and was independent of acetylation of sialic acid residues. To map the neutralizing epitopes on VP8*, we developed a panel of monoclonal antibodies inhibiting hemagglutination of VP8* protein. One of these antibodies (2E8) was used to generate a mutant virus resistant to neutralization by the monoclonal antibody. The antibody escape mutant was found to contain one point mutation at amino acid position 116 (Glu - > Asp). To investigate the effect of this escape mutation on the cellular binding and hemagglutination activities, the VP8* gene of the escape mutant was expressed in bacteria, and its functional activities were compared with those of the parental strains. To understand the mechanism of virus neutralization by 2E8, radiolabeled BRV was incubated with 2E8 and the complex purified by centrifugation through a CsCl gradient. The distribution of radioactivity was analysed, and the peak fractions were observed by electron microscopy. The radioactivity profile of the virus was altered, and its structural integrity destroyed by 2E8. The virus was not affected by non-neutralizing monoclonal antibodies when compared to the radioactivity profile by BRV alone. This result suggests that 2E8 binds to BRV VP8*, affecting the stability of the particle, and may thereby neutralize virus infectivity. To examine if this antigen could induce neutralizing antibody responses in vivo, different species of animals were immunized with rVP8*. (Abstract shortened by UMI.)
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Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology