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. 2024 Jan 25;14(3):389.
doi: 10.3390/ani14030389.

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

Gyu-Nam Park  1 , SeEun Choe  1 , Sok Song  1 , Ki-Sun Kim  1 , Jihye Shin  1 , Byung-Hyun An  2 , Soo Hyun Moon  1 , Bang-Hun Hyun  1 , Dong-Jun An  1
Affiliations

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

Gyu-Nam Park et al. Animals (Basel). .

Abstract

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.

Keywords: BCoV; CPE; calf; mutation; vaccine.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Non-CPE and CPE observed after inoculation of the BCoV KBR-1 strain onto HRT-18 and MDBK cells. BF (A) and IFA (B) images obtained at 4 days post-KBR-1 inoculation, and BF (C) and IFA (D) images obtained at 4 days post-mock inoculation, into HRT-18 cells. BF (E) and IFA (F) images obtained at 4 days post-inoculation of the KBR-1 strain, and BF (G) and IFA (H) images obtained at 4 days post-mock inoculation, into MDBK cells. BF = bright field; IFA = indirect fluorescence assay.
Figure 2
Figure 2
Maximum-likelihood phylogenetic tree (LogL = −14799.04) based on the complete S gene nucleotide sequences of 106 BCoV reference strains detected in Asian, North American, and European countries, including the 12 KBR-1 strains passaged in cells in this study. The phylogenetic tree was constructed using MEGA X software (the Tamura–Nei model and the nearest-neighbor interchange method). The blue box marks the 12KBR-1 strains.
See this image and copyright information in PMC

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