Plaque Variations in Clinical Isolates of Bovine Coronavirus

Bovine coronavirus (BCV) causes enterocolitis and respiratory disease in young calves and may cause chronic diarrhea (winter dysentery) in adult cattle. To understand the molecular basis of tissue tropism and virulence, and to study biological differences between clinical isolates, it is important to plaque purify BCV. Plaque assay can also be used to study the effect(s) of antiviral drugs, such as hygromycin B, on BCV replication in vitro. Moreover, there is an urgent need to select better vaccine candidates and develop an effective modified live virus vaccine against BCV, and candidate vaccine isolates must be plaque purified for characterization. Because isolates of BCV are obtained from fecal samples that contain a wide variety of bacteria and viruses, plaque purification is essential. This paper describes the development of a plaque assay to study plaque variation in recent isolates of BCV (1993-1994) and to study the antiviral effects of hygromycin B on BCV in vitro. Human rectal tumor-18 (HRT-18) cells were plated in 6-mm tissue culture dishes and grown in minimum essential medium (MEM) with 10% fetal calf serum, L-glutamine, and antibiotics (Kapil S, Donnelly C: 1994, Abstr Proc 75th Conf Res Workers Anim Dis, P77). After 3-4 days, confluent monolayers were washed with calciumand magnesium-free phosphate-buffered saline (CMF-PBS). BCV isolates were obtained from calves with a history of diarrhea. Approximately 250 μl of virus dilutions (10 -10) was added to these plates. For BCV adsorption, the plates were hand-rocked every 5-10 minutes for 1 hour. Monolayers were washed with CMF-PBS and overlaid with 1% agarose in MEM containing trypsin (5 μg/ml) and pancreatin (5 μg/ml). To study the effect of hygromycin B on BCV replication the drug was incorporated in the agarose at concentrations of 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM. Plates were incubated in an inverted position in a humid chamber at 37 C for 3-5 days. To count and measure the plaques, the plates were held against a bright source of light. Since our protocol gave clearly visible plaques, it was not necessary to stain the plates. Viral titers were calculated on the basis of dilutions that gave about 2530 isolated plaques/well. To confirm the identities of the plaques, the monolayers were fixed with 5 ml of 10% neutral buffered formalin per plate. In preliminary experiments, we compared 100% methanol, 100% ethanol, 5% glutaralde-