Date of Award

1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Johannes Storz

Abstract

Human rectal tumor-18 (HRT-18) cell clones 3F3, 3E3, D2, and 4B3 exhibited differences in cellular morphology in Giemsa-stained cultures and developing monolayers. Differences were evident in growth kinetics and plating efficiency of each clone. The clones produced colonies in soft agar, demonstrating anchorage independence. Cytopathic expression (CPE) including cytoplasmic vacuolization and cell fusion occurred in BCV-L9-infected clones 3F3, D2, and 3E3. Cell fusion was inapparent in clone 4B3. Bovine coronavirus strain L9 (BCV-L9) and 5 wild-type isolates replicated in HRT-18 cells, inducing cell fusion. Strain L9, exclusively, replicated in D2BFS cells, requiring trypsin to induce cell fusion. Strain L9 produced plaques in the HRT-18 clones, but the ease of plaque formation and plaque morphology was host cell dependent. Host cell-dependent plaque formation was demonstrated by wild-type BCV strains, and plaque morphology was strain dependent. The intensity of trypsin enhancement of CPE and plaque development depended on the virus strain and host cell. Trypsin greatly enhanced the infectivity of BCV-L9 in D2BFS and HRT-18 cells, and to lesser extents in clones 3F3, 3E3, and D2. Trypsin-enhanced infectivity was not detected in clone 4B3. The infectivity of strain LY-138 in HRT-18 cells was slightly enhanced by trypsin. Eleven structural proteins of BCV-L9 were detected by immunoblotting, including 185, 160, 140, 125, 110, 100, 52, 46, 37, 31-34, and 26-28 Kd species. Under reducing conditions 185, 140, and 100 Kd species migrated as 190, 65, and 95 Kd forms. Silver-stained proteins of 18, 20, and 23 Kd were reduced to a 20-23 Kd cluster. Host cell-dependent differences in the protein profile of L9 were detected. A 62 Kd protein was specific to L9 (D2BFS with trypsin). L9 (4B3) and L9 (D2) lacked the 46 and 110 Kd bands, respectively. Cleavage of the 185 Kd protein, an increase in the 100 Kd band, and conversion of the 31-34 Kd cluster to a 34 Kd band occurred when BCV-L9 was propagated in D2BFS cells with trypsin. Trypsin converted the reduced, 95 Kd protein found in L9 (HRT-18) to 90 Kd, and the 20-23 Kd cluster was altered to 19-23 Kd. In vitro trypsin treatment of L9 (3F3) resulted in cleavage of the 46 Kd molecule and the appearance of a 25 Kd species.

Pages

161

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