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

Simple Summary Diarrheal diseases in calves cause economic losses in countries worldwide. In particular, bovine coronavirus, the main cause of diarrhea in calves, results in high economic losses for cow farmers. In South Korea, calf diarrhea, which affects newborn calves, and winter dysentery, which occurs in adult cows during the winter, are detected continuously. The vaccine strain (BC94) used in South Korea belongs to the GI type; however, a phylogenetic analysis revealed that all of the prevalent circulating strains belong to the GIIa type. Therefore, we attempted to develop a live attenuated BCoV vaccine candidate that targets recent prevalent strains. 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.

BCoV is an important pathogen that causes not only neonatal calf diarrhea (NCD) [2], but also winter dysentery (WD) in adult cattle [3] and respiratory disease in cattle of all ages [4,5].BCoV particles comprise four major structural proteins: the membrane (M) glycoprotein, an envelope (E) protein, a spike (S) glycoprotein, and the hemagglutinin-esterase (HE) glycoprotein [6].In particular, the S glycoprotein possesses domains responsible for receptor binding and the induction of neutralizing antibodies, which have been exploited for the molecular characterization of isolates [7].Additionally, the S glycoprotein Animals 2024, 14, 389 2 of 10 comprises two subunits: S1 (N-terminal half) and S2 (C-terminal half).Of these, S1 is a globular subunit responsible for virus binding to host cell receptors [8], the induction of neutralizing antibodies [9], and hemagglutinin activity [10].The S1 hypervariable region is also useful for studying the variability and evolution of BCoV [11,12].The S1 sequence is diverse, and mutations in this region are associated with changes in antigenicity and viral pathogenicity [13].
WD and NCD have occurred in Korea since the early 2000s [14,15].Additionally, animal experiments confirmed that BCV, the cause of WD, can induce pathogenic effects in both the digestive and respiratory systems of calves [15].Recent studies show that BCoVs detected in the diarrheal feces of Korean calves belong to the GIIa genotype, suggesting that there is an antigenic difference from the currently used BC94 (G1 genotype) vaccine strain [16,17].Thus, it unclear whether the classical BCoV vaccine protects against the recently prevalent GIIa genotype; this is particularly important because as BCoV continues to evolve, new genetic types are being formed and the genes are diversifying.Therefore, it is necessary to develop vaccines that are specific for various BCoV genotypes.
The purpose of this study was to develop a novel live attenuated vaccine candidate through continuous passage of the BCoV isolate prevalent in South Korea, and to conduct a comparative analysis of the S gene.Additionally, a BCoV isolate (KBR-1-p10) and the attenuated candidate strain (KBR-1-p120) were administered orally to calves, after which diarrhea scores, virus shedding, and the presence of viruses in organs were evaluated.

Cell Passage and Indirect Immunofluorescence Assay
This study used a KBR-1 strain isolated from a calf with diarrhea in the Boryeong region (located approximately 150 km south of Seoul) of South Korea [17].The KBR-1 strain was propagated via inoculation at a multiplicity of infection (MOI) of 0.1 onto monolayers of human rectal tumor (HRT-18) cells cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) containing 5 µg/mL of trypsin (Gibco, Grand Island, NY, USA), followed by incubation at 37 • C/5% CO 2 for 4 days.Additionally, to increase the titer of the KBR-1 strain, it was inoculated (MOI = 0.1) onto Madin-Darby bovine kidney (MDBK) cells cultured in α-MEM (Gibco, Grand Island, NY, USA) containing 5 µg/mL of trypsin (Gibco, Grand Island, NY, USA), followed by incubation at 37 • C/5% CO 2 for 4 days to observe cytopathic effects (CPEs).At 4 days post-inoculation of the KBR-1 strain onto HRT-18 or MDBK cells, virus proliferation was confirmed in an indirect immunofluorescence assay (IFA).Briefly, at 4 days post-KBR-1 inoculation, cells were fixed in 80% cold acetone and then reacted for 1 h with a monoclonal antibody specific for BCoV (Absolute Antibody Ltd, Cat no.EGO012, Wilton Centre Redcar, UK).Additionally, after reacting cells for 1 h with FITC-labeled anti-mouse IgG (H + L) adsorbed on human serum (SeraCare Life Sciences, Inc., Cat no.5230-0307, Milford, MA, USA), fluorescence was observed under a fluorescence microscope.

Phylogenetic Analysis of BCoV
The complete S gene sequences of 12 KBR-1 strains of BCoV, obtained after inoculation onto HRT-18 cells and MDBK cells, were used for phylogenetic analysis.Additionally, the complete S gene sequences of 94 reference BCoV strains were obtained from the National Center for Biotechnology Information (NCBI) GenBank database.Multiple-nucleotide sequence alignments of all 106 BCoV strains were carried out in the CLUSTAL X alignment program (ver.2.1.)[18], and were analyzed phylogenetically using the MEGA X software [19] using the maximum-likelihood (ML) method, the Tamura-Nei model, and bootstrap analysis (n = 1000).The ML tree was constructed using rates among sites (gamma distributed with invariant sites (G + I)) and the ML heuristic method (nearest-neighbor interchange (NNI)).

Comparison of the S Genes of Passaged BCoV Strains
After the inoculation of the BCoV KBR-1 strain into HRT-18 and MDBK cells, total RNA was extracted using the RNeasy mini kit (Qiagen, Cat.No. 74104, Maryland, MD, USA), and cDNA was prepared using the Helix Cript™ Easy cDNA Synthesis Kit (NanoHelix, Daejeon, Republic of Korea).We conducted a PCR of the complete S gene (4092 bp), using six previously published oligo primer sets [20], with amplified fragments of 920 bp, 769 bp, 828 bp, 872 bp, 916 bp, and 653 bp.The six PCR products were sequenced at the Cosmo Genetech Institute (Daejeon, Republic of Korea) using an ABI Prism 3730xl DNA sequencer to generate the complete S gene sequence.Comparative analyses of the complete BCoV S genes were carried out via multiple sequence alignments using Bio Edit Sequence Alignment Editor (version 7.2).

Calf Experiments
To investigate the pathogenicity of the KBR-1-p120 strain (passaged in MDBK cells) and the KBR-1-p10 strain (passaged in HRT-18 cells), 7-day-old calves (negative for BCoV antigens in real-time PCR [21] and negative for antibodies in a serum neutralization antibody test [17]) that did not consume colostrum were assigned to two groups (G1 and G4).Two calves in the G1 group received an oral inoculation of KBR-1-p120 at 10 7.0 TCID 50 /mL per dose.The G4 group received KBR-1-p10 (concentrated through density gradient ultracentrifugation due to a low titer (10 3.5−4.0TCID 50 /mL)) via an oral inoculation of 10 5.0 TCID 50 /mL per dose.Clinical symptoms (appetite, activity, diarrhea, and nasal) were scored for 7 days after oral inoculation of the KBR-1-p10 and KBR-1-p120 strains.Clinical symptoms were scored as follows: appetite (normal appetite: 0; abnormal appetite: 1); activity (normal activity: 0; abnormal activity: 1); nasal symptoms (normal nasal: 0; rhinorrhea: 1); and diarrhea (no diarrhea: 0; mild diarrhea: 1; moderate diarrhea: 2; and severe diarrhea: 3).Additionally, fecal and nasal samples were collected daily for 7 days post-inoculation (DPI).These samples were collected using sterilized cotton swabs, placed in a liquid transport medium, vortexed, and stored in a deep freezer at −70 • C until realtime PCR (RT-PCR).BCoV RNA was detected via RT-PCR to obtain cycle threshold (ct) values [21].To examine the pathogenic reversion of the KBR-1-p120 strain, the two calves in group G1 (inoculated with the KBR-1-p120 strain) were autopsied at 7 DPI, and the small intestines were emulsified.The emulsion (5 mL per dose) was then inoculated into two 7-day-old calves (group G2).These two calves were then autopsied 7 days later, and their small intestines were emulsified; 5 mL of this emulsion was then administered to two 7-day-old calves (group G3), and these calves were also autopsied at 7 DPI.

Detection of BCoV RNA via RT-PCR
Fecal and nasal samples from all calves used in the above experiment, as well as samples of their colon, duodenum, jejunum, and ileum, were subjected to RT-PCR as described previously [21].RT-PCR targeting the N gene of BCoV was conducted using forward primer CTA GTA ACC AGG CTG ATG TCA ATA CC, reverse primer GGC GGA AAC CTA GTC GGA ATA, and probe FAM-CGC CTG ACA TTC TCG ATC-MGB [20].RT-PCR was performed using the CFX Opus 96 model (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the following cycling conditions: reverse transcription at 45 • C for 30 min; activation of DNA polymerase at 95 • C for 10 min; 40 cycles of denaturation at 94 • C for 15 s; and annealing/elongation at 60 • C for 60 s.

Changes in Cell Morphology Induced by the KBR-1 Strain
The BCoV KBR-1 strain was isolated successfully after inoculating susceptible HRT-18 cells, and virus growth was confirmed in an IFA [17]; however, no CPE was observed under a bright field (BF) microscope, although some trypsin-induced damage was observed (Figure 1).A previous study reported that the KBR-1 strain grew to 10 3.0 TCID 50 /mL at passage five, 10 4.2 TCID 50 /mL at passage 20, and 10 6.2 TCID 50 /mL at passage 40, in HRT-18 cells [17]; however, the maximum proliferation of the KBR-1 strain observed in the present study was <10 6.1 TCID 50 /mL (10 6.1 TCID 50 /mL at passage 50, 10 5.8 TCID 50 /mL at passage 60, 10 5.6 TCID 50 /mL at passage 70, and 10 5.5 TCID 50 /mL at passage 79).To increase the virus titer, we inoculated the KBR-1 strain continuously onto susceptible MDBK cells from passage 80 to passage 120; the virus titer then increased to 10 7.7 TCID 50 /mL (10 7.3 TCID 50 /mL at passage 90, 10 7.6 TCID 50 /mL at passage 100, 10 7.7 TCID 50 /mL at passage 110, and 10 7.5 TCID 50 /mL at passage 120).In addition, the KBR-1 strain exhibited CPEs in susceptible MDBK cells (Figure 1), which is consistent with the IFA results (Figure 1) obtained using a BCoV-specific fluorescent antibody.A recent BCoV isolation study inoculated MDBK cells with calf diarrhea samples obtained from the central part of Oromia, Ethiopia, and also demonstrated CPEs [22].The MDBK cells began clumping after 24 h, and appeared thin and round after 48 h; the majority of the monolayer detached after 72 h [22].Thus, MDBK cells show CPEs and allow the virus to propagate to high titers.Therefore, they are considered highly sensitive to BCoV infection, making them a useful tool for virus isolation.
under a bright field (BF) microscope, although some trypsin-induced damage was o served (Figure 1).A previous study reported that the KBR-1 strain grew to 10 3.0 TCID50/m at passage five, 10 4.2 TCID50/mL at passage 20, and 10 6.2 TCID50/mL at passage 40, in HR 18 cells [17]; however, the maximum proliferation of the KBR-1 strain observed in the pr sent study was <10 6.1 TCID50/mL (10 6.1 TCID50/mL at passage 50, 10 5.8 TCID50/mL at passa 60, 10 5.6 TCID50/mL at passage 70, and 10 5.5 TCID50/mL at passage 79).To increase the vir titer, we inoculated the KBR-1 strain continuously onto susceptible MDBK cells from pa sage 80 to passage 120; the virus titer then increased to 10 7.7 TCID50/mL (10 7.3 TCID50/m at passage 90, 10 7.6 TCID50/mL at passage 100, 10 7.7 TCID50/mL at passage 110, and 10 TCID50/mL at passage 120).In addition, the KBR-1 strain exhibited CPEs in susceptib MDBK cells (Figure 1), which is consistent with the IFA results (Figure 1) obtained usin a BCoV-specific fluorescent antibody.A recent BCoV isolation study inoculated MDB cells with calf diarrhea samples obtained from the central part of Oromia, Ethiopia, an also demonstrated CPEs [22].The MDBK cells began clumping after 24 h, and appear thin and round after 48 h; the majority of the monolayer detached after 72 h [22].Thu MDBK cells show CPEs and allow the virus to propagate to high titers.Therefore, th are considered highly sensitive to BCoV infection, making them a useful tool for vir isolation.
* No. P: number of passages in cell culture.

Clinical Signs/Symptoms and Virus Shedding by Infected Calves
The clinical scores for the two calves inoculated with the KBR-1-p10 strain (G4) were one (weak) and two (moderate) between 4 and 7 days post-inoculation.In addition, their appetite and activity decreased over the same time period (days 4-7) (Table 2).By contrast, calves in G1 (KBR-1-p120), G2 (inoculated with calf small intestine emulsion from G1), and G3 (inoculated with calf small intestine emulsion from G2) showed no symptoms of diarrhea or runny nose (Table 2).Their appetite and activity were normal (Table 2).The RT-PCR to detect BCoV RNA in nasal samples obtained at 7 days post-inoculation revealed that it was absent from some calf samples (Table 2).A previous study showed that WD-type BCoV can infect both the digestive and respiratory tracts of calves [15]; however, the KBR-1 strain used in the present study (an NCD-type BCoV) is believed to infect only the digestive tract.BCoV RNA from feces samples collected from G4 showed a ct value of 24.8-33.9for calf G4-1 and 25.1-34.2for calf G4-2 (Table 2).Calf (G1-1) in the G1 group had BCoV RNA in its feces, with a ct value of 37.6-38.1,whereas calf G1-2 had a ct value of 36.2-37.5.However, a previous paper that performed RT-PCR using the primers and probes used in the present study defined a positive RT-PCR ct value < 35 [28].Therefore, in the pathogenicity reversal experiment using the KBR-1-p120 strain, the ct values detected in samples from calves in the G1 group were almost negative.Additionally, the ct values in calves from the G2 and G3 groups were negative (Table 2).These data infer that the KBR-1-p120 strain was attenuated.

Organs from Calves Inoculated with BCoV
The two calves in G4 (inoculated with the KBR-1-p10 strain) were autopsied 7 days post-inoculation to detect BCoV RNA in the duodenum, ileum, and jejunum (Table 3).The ct value in the jejunum and ileum of G1-1 (inoculated with the KBR-1-p120 strain) was >35; the ct value in the ileum of calf G1-2 was also high (36.9)(Table 3).However, it was difficult to detect BCoV (KBR-1-p120) in the organs of the G1 group calves because the ct values were so high.A previous paper showed that when the mock group was challenged with virulent BCoV, the virus was retained in the organs, especially the duodenum, jejunum, and

Figure 1 .
Figure 1.Non-CPE and CPE observed after inoculation of the BCoV KBR-1 strain onto HRT-18 a MDBK cells.BF (A) and IFA (B) images obtained at 4 days post-KBR-1 inoculation, and BF (C) a IFA (D) images obtained at 4 days post-mock inoculation, into HRT-18 cells.BF (E) and IFA images obtained at 4 days post-inoculation of the KBR-1 strain, and BF (G) and IFA (H) imag obtained at 4 days post-mock inoculation, into MDBK cells.BF = bright field; IFA = indirect fluor cence assay.

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.

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.

Table 1 .
Amino acid sequences of the spike gene of bovine coronavirus KBR-1 strains.

Table 2 .
Clinical scores and virus shedding (up to 7 days post-BCoV inoculation).