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Document Type

Thesis - University Access Only

Award Date

1992

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

David A. Benfield

Abstract

Human Rectal Tumor (HRT) cells are permissive for in vitro amplification of bovine enteric coronavirus (BECV). The objectives of this study were two-fold. The first was to determine reliable methods for obtaining maximum BECV production in vitro, and ·the second, to identify and characterize cellular receptors that allow BECV to bind to and enter HRT cells. In the initial study the effects of cell age, adsorption temperature and time, and pH of adsorption media on the infectivity of BECV was assessed with a fluorescent focus assay. This study indicated that optimum BECV infectivity titers were obtained if virus was allowed to adsorb to 5 day old HRT cells for 150 minutes at 37°C with media adjusted to pH 7.5. Three glycoproteins (12, 17, 32 kDa) that bind BECV were identified in solubilized membrane preparations of HRT cells. Binding of BECV to these glycoproteins was demonstrated by a direct virus binding assay (DVBA), which uses [35S]BECV as a probe, and a virus overlay protein blot assay (VOPBA), which has been as previously described for the murine coronavirus, mouse hepatitis virus (4). The specificity of these binding glycoproteins for [35S]BECV was demonstrated by the absence of binding when [35S]mock virus preparations replaced BECV in the assay and binding of radiolabeled BECV was blocked in the presence of a 10-fold excess of non-labeled virus. In the VOPBA, binding to the three glycoproteins was observed only when virus was present in the assay. The BECV-binding glycoproteins were not detected on membranes of non-permissive cell lines, MA-104, baby hamster kidney and swine testis, indicating that the presence of these binding glycoproteins may define the susceptibility of a cell line to BECV infection. Pre-treatment of the HRT cell membranes with sodium metaperiodate destroyed all binding activity, indicating that the interaction between BECV and the three glycoproteins requires the carbohydrate moiety. However, sialic acid was not required for binding of BECV, because pre-treatment of HRT cell membranes with neuraminidase had no effect on the binding of BECV to the glycoproteins. These data suggest the three glycoproteins on the surface of HRT cells may determine susceptibility to BECV infection by mediating the binding of BECV to susceptible cells.

Library of Congress Subject Headings

Cattle -- Virus diseases
Viruses -- Receptors
Coronavirus infections

Format

application/pdf

Publisher

South Dakota State University

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