Isolation and trypsin-enhanced propagation of turkey enteric (bluecomb) coronaviruses in a continuous human rectal adenocarcinoma cell line

Turkey enteric coronavirus (TCV) from intestinal contents of diarrheal poults was isolated and serially propagated in HRT-18 cells, an established cell line derived from a human rectal adenocarcinoma. In these cells, TCV induced cytopathic changes, including polykaryocytosis, which depended on trypsin in the medium and incubation at 41 C. Viral antigens could be demonstrated in the cytoplasm by immunofluorescence, and extracellular virus was detected by an ELISA and negative electron microscopy. The cell-free virus had characteristics of TCV: shape, surface projections, buoyant density of 1.18 to 1.20 g/ml in sucrose, and hemagglutination of rat RBC. The one-step growth curve was complete by postinoculation hours 14 to 16, and maximal titers reached 9 to 9.5 log10 TCID50/ml during 5 passages, after which the titer remained stable. Electron microscopic examination of infected cell monolayers revealed budding of typical coronavirus particles through intracytoplasmic membranes and accumulation of complete virus in cytoplasmic vesicles. Late in the infection, aggregated progeny vial particles were detected near the outer surface of infected cells. One-day-old turkey poults inoculated orally with tissue culture-adapted TCV isolates developed mild to severe diarrhea.