Characterization and Spike Gene Analysis of a Candidate Attenuated Live Bovine Coronavirus Vaccine

The bovine coronavirus (BCoV) KBR-1 strain, obtained from calf diarrhea samples collected in 2017, belongs to group GIIa. To attenuate this strain, it was subcultured continuously (up to 79 times) in HRT-18 cells, followed by 80-120 passages in MDBK cells. The KBR-1-p120 strain harvested from MDBK cells at passage 120 harbored 13 amino acid mutations in the spike gene. Additionally, the KBR-1-p120 strain showed a high viral titer and cytopathogenic effects in MDBK cells. Seven-day-old calves (negative for BCoV antigen and antibodies) that did not consume colostrum were orally inoculated with the attenuated candidate strain (KBR-1-p120), or with KBR-1 passaged 10 times (KBR-1-p10) in HRT-18 cells. Calves inoculated with KBR-1-p10 had a low diarrhea score, and BCoV RNA was detected at 3-7 days post-inoculation (DPI). The virus was also present in the duodenum, jejunum, and ileum at autopsy; however, calves inoculated with KBR-1-p120 had low levels of BCoV RNA in feces at 4-6 DPI, and no diarrhea. In addition, an extremely small amount of BCoV RNA was present in the jejunum and ileum at autopsy. The small intestines of calves inoculated with KBR-1-p120 were emulsified and used to infect calves two more times, but pathogenicity was not recovered. Therefore, the KBR-1-p120 strain has potential as a live vaccine candidate.