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Adaptation of an isolate of bovine enteritic coronavirus to serial growth in cell culture and characterization of the virus



Adaptation of an isolate of bovine enteritic coronavirus to serial growth in cell culture and characterization of the virus



Annales de Recherches Veterinaires. Annals of Veterinary Research 12(3): 243-251



Coronaviruses are known to behave as pathogens in a variety of animal species. Their multiplication in the differentiated enterocytes of the intestinal tract induces a severe diarrheic syndrome which may lead to death. To study these viruses and to progress in the development of prophylactic and diagnostic methods, it is essential to succeed in adapting the agents to serial growth in cell culture. In spite of several attempts adaptation to primary cell cultures could be obtained in the past only for two isolates. The present work describes the methodology which enabled us to succeed in the adaptation of a bovine enteric coronavirus isolated in France to primary calf kidney cells, and reports results on the characteristics of this virus. Adaptation to serial growth was realized at 34 degrees C in the following way: 3 passages each lasting 8 to 10 days followed by 16 passages each lasting 6 days. From the 20th passage on the duration of incubation was reduced to 4 days. Virus growth was checked using an indirect immunofluorescence test. During the first passages only a few dispersed positive cells could be observed but later on small foci of immunofluorescent cells did appear. By the 35th passage infective titers did not exceed 1 x 10(5) TCID50/ml and cytopathic effects were always very discrete. When the susceptibility of different cell-types to this coronavirus strain (G110) was checked we found that only bovine cells and the human HRT18 cell-line were able to replicate the virus. In HRT18 cells titers as high as 5 x 10(7) TCID50/ml could be obtained at the first passage of G110 strain, previously adapted to primary calf kidney cells. As regards the antigenic properties of G110 virus no difference could be established between this strain and the Nebraska isolate (NCDCV) neither by indirect immunofluorescence test nor by indirect neutralization test. The G110 strain agglutinates rat and mouse erythrocytes. The density of purified virions in mouse gradients is 1.19 and the morphology of the particles is characteristic of a coronavirus.

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Accession: 004673734

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PMID: 6284011


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