Cultivation Of Bovine Coronavirus Attenuated Strains And Immunogenicity In BALB/c Mice

Bovine coronavirus (bovine coronavirus, BCoV) was one of the most important pathogenic that cause calfdiarrhea, respiratory diseases and winter dysentery in adult cattle. The distinct clinical syndromes in calf werecalf diarrhea, the adult cattle winter diarrhea (sometimes accompanied by blood and mucus) and so on, Whichresulted in severe economic losses to the cattle industry all over the world..In this article, the BCoV-DQ strain was activated, proliferated in HRT-18cells and purified virus strainsobtained by plaque assay. BCoV-DQ strain was attenuated by serial passages in Vero cell lines for25generations. TCID50/0.1ml of BCoV was downward tendency alone with passages in vero cells, which isdown over3orders of magnitude from10-6.875to10-3.50..But The TCID50were stable between10-3~10-4ingeneration15~25. The results of the RT-PCR and TCID50showed that the BCoV-Vero25were stable andattenuated.The virus culture fluid intragastriclly, intranasally, abdominal infected6to8weeks of age femaleBALB/c mice with100TCID50～700TCID50BCoV doses. The mice were observed the clinical changes,changes in body weight, pathological changes of contrast necropsy and histopathological changes. All of theseillustrate that300TCID50BCoV-DQ infected BALB/c mice can cause significant clinical symptoms andpathological changes.The BCoV-Vero25infected animals by300TCID50in different ways, without causing significant damagein experimental animals. The no revertion to virulence was shown on3passages after BCoV-Vero25wasserial passaged three times in mice. the RT-PCR confirmed the attenuated strain virus survived in mice and norevertion to virulence.BALB/c mice as an animal model were inoculated orally with BCoV-Vero25. Specific IgG antibody inthe serum and specific immunoglobulin A (sIgA) antibody in the lung, intestines, vagina fluid and feces ofimmunized mice were detected by indirect ELISA. T-cell proliferation responses were investigated by MTTtest. The mice orally immunized with BCoV-Vero25were challenged with BCoV-DQ. The ELISA showedthat the mice immunized with BCoV-Vero25could produce remarkable specific sIgA level in the fecessamples and lavage fluids samples. More than90%of the vaccinated mice were protected by challenged with300TCID50BCoV-DQ. All of the results indicated that the attenuated strain has a protective effect in mice andmay be a candidate to protect calf against BCoV-DQ infection.