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Bovine coronavirus isolation and cultivation in continuous cell lines

Bovine coronavirus isolation and cultivation in continuous cell lines

American Journal of Veterinary Research 41(1): 30-38

Vero [African green monkey kidney], Madin-Darby bovine kidney, and porcine kidney-15 continuous cell lines were satisfactory for the isolation and multiplication of the Nebraska calf diarrhea coronavirus (NCDC), and bovine coronaviruses isolated from affected calves with scours in Quebec dairy and beef herds. A readily recognizable cytopathic effect (CPE) was produced in these cell cultures as early as the 2nd or 3rd passage. The viruses could also be serially propagated in these cell cultures, and EM examination of infected cells showed characteristic coronavirus particles identical to those found in intestinal contents of diarrheal calves. Viral antigens could also be demonstrated by immunofluorescent staining, and viral yield varied between 106-107 median tissue culture infective dose/ml after 10 to 15 passages. A CPE was also produced in fetal bovine kidney cells, but it was difficult to recognize, and CPE was not produced in several other continuous cell lines tested or in primary fetal lamb kidney cells. Syncytia were not detected in susceptible cell cultures. It was also demonstrated that the infection of Vero cells by NCDC could be managed culturally in such a way as to produce a maximum of the viral production capacity whether by acting on the cell adsorption properties or on the cell metabolism. The addition of heat-inactivated bovine fetal serum without .gamma.-globulins to the maintenance medium (up to 5%) did not inhibit the growth of the virus in these cells, whereas the pH of inoculation and incubation media affected it significantly. Cell-sheet treatment with dactinomycin (up to 0.05 .mu.g/ml), trypsin (up to 20 .mu.g/ml), DEAE-dextran (up to 25 .mu.g/ml) and hypertonic sodium significantly increased the production of NCDC or appearance of viral cytopathogenicity or both.

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Accession: 000606903

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PMID: 6767425

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