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.

Efficacy of an orally administered classical swine fever live marker vaccine (Flc-LOM-BErns strain) in pigs.

In several countries, classical swine fever (CSF) has not been detected in domestic pigs, but has been detected in wild boars, making the disease difficult to control. To overcome this problem, we inoculated pigs with a CSF live marker vaccine (Flc-LOM-BErns strain), which has "distinguish infection from vaccinated animals (DIVA)" function, to determine whether it is suitable as an oral vaccine specifically for wild boars. Pigs inoculated intramuscularly or orally with the Flc-LOM-BErns vaccine were challenged 2 or 4 weeks later, respectively, with virulent CSFV. Pigs administered the oral Flc-LOM-BErns strain (105.0 and 6.0 TCID50/dose), and those vaccinated intramuscularly (103.0 TCID50/dose), had normal numbers of leukocytes and normal body temperature. Also, they generated protective neutralizing antibodies and anti-BVDV Erns antibodies. In addition, all pigs in these groups survived, with no CSFV RNA detected in feces, spleen, or other organs. Thus, the Flc-LOM-BErns vaccine shows excellent safety and efficacy, while having DIVA function and suitability for oral inoculation.

Spike Gene Analysis and Prevalence of Porcine Epidemic Diarrhea Virus from Pigs in South Korea: 2013-2022.

From late 2013-2022, 1131 cases of porcine epidemic diarrhea (PED) were reported to the Korean Animal Health Integrated System (KAHIS). There were four major outbreaks from winter to spring (2013-2014, 2017-2018, 2018-2019, and 2021-2022), with the main outbreaks occurring in Chungnam (CN), Jeonbuk (JB), and Jeju (JJ). Analysis of the complete spike (S) gene of 140/1131 KAHIS PEDV cases nationwide confirmed that 139 belonged to the G2b genotype and 1 to the G2a genotype. Among them, two strains (K17GG1 and K17GB3) were similar to an S INDEL isolated in the United States (strain OH851), and 12 strains had deletions (nucleotides (nt) 3-99) or insertions (12 nt) within the S gene. PEDVs in JJ formed a regionally independent cluster. The substitution rates (substitutions/site/year) were as follows: 1.5952 × 10-3 in CN, 1.8065 × 10-3 in JB, and 1.5113 × 10-3 in JJ. A Bayesian skyline plot showed that the effective population size of PEDs in JJ fell from 2013-2022, whereas in CN and JB it was maintained. Genotyping of 340 Korean PEDV strains, including the 140 PEDVs in this study and 200 Korean reference strains from GenBank, revealed that only the highly pathogenic non-INDEL type (G2b) was dominant from 2020 onwards. Therefore, it is predicted that the incidence of PED will be maintained by the G2b (non-INDEL) genotype.

Phylogenetic analysis of swine influenza A (H1N2) viruses isolated in Jinju City, Republic of Korea.

Genomic sequences of the swine influenza A (H1N2) viruses "A/Swine/South Korea/GN-1/2018" and "A/Swine/South Korea/GNJJ/2020" sampled from Jinju City, Republic of Korea, are reported here. The sequences of these viruses were 99% similar. These included eight genes from each of the H3N2pM, A(H1N1)2009pdm, and North American swine lineages.

Assessment of Equine Influenza Virus Status in the Republic of Korea from 2020 to 2022.

Equine influenza virus (EIV) causes acute respiratory disease in horses and belongs to the influenza A virus family Orthomyxoviridae, genus Orthomyxovirus. This virus may have severe financial implications for the horse industry owing to its highly contagious nature and rapid transmission. In the Republic of Korea, vaccination against EIV has been practiced with the active involvement of the Korea Racing Authority since 1974. In this study, we monitored the viral RNA for EIV using PCR, as well as the antibody levels against 'A/equine/South Africa/4/03 (H3N8, clade 1)', from 2020 to 2022. EIV was not detected using RT-PCR. The seropositivity rates detected using a hemagglutination inhibition assay were 90.3% in 2020, 96.7% in 2021, and 91.8% in 2022. The geometric mean of antibody titer (GMT) was 83.4 in 2020, 135.7 in 2021, and 95.6 in 2022. Yearlings and two-year-olds in training exhibited lower positive rates (59.1% in 2020, 38.9% in 2021, and 44.1% in 2022) than the average. These younger horses may require more attention for vaccination and vaccine responses against EIV. Continuous surveillance of EIV should be performed to monitor the prevalence and spread of this disease.

Safety and Immunogenicity of Chimeric Pestivirus KD26_E2LOM in Piglets and Calves.

A chimeric pestivirus (KD26_E2LOM) was prepared by inserting the E2 gene of the classical swine fever virus (CSFV) LOM strain into the backbone of the bovine viral diarrhea virus (BVDV) KD26 strain. KD26_E2LOM was obtained by transfecting the cDNA pACKD26_E2LOM into PK-15 cells. KD26_E2LOM chimeric pestivirus proliferated to titers of 106.5 TCID50/mL and 108.0 TCID50/mL at 96 h post-inoculation into PK-15 cells or MDBK cells, respectively. It also reacted with antibodies specific for CSFV E2 and BVDV Erns, but not with an anti-BVDV E2 antibody. Piglets (55-60 days old) inoculated with a high dose (107.0 TCID50/mL) of KD26_E2LOM produced high levels of CSFV E2 antibodies. In addition, no co-habiting pigs were infected with KD26_E2LOM; however, some inoculated pigs excreted the virus, and the virus was detected in some organs. When pregnant sows were inoculated during the first trimester (55-60 days) with a high dose (107.0 TCID50/mL) of KD26_E2LOM, anti-CSFV E2 antibodies were produced at high levels; chimeric pestivirus was detected in one fetus and in the ileum of one sow. When 5-day-old calves that did not consume colostrum received a high dose (107.0 TCID50/mL) of KD26_E2LOM, one calf secreted the virus in both feces and nasal fluid on Day 2. A high dose of KD26_E2LOM does not induce specific clinical signs in most animals, does not spread from animal to animal, and generates CSFV E2 antibodies with DVIA functions. Therefore, chimeric pestivirus KD26_E2LOM is a potential CSFV live marker vaccine.

Selection and Evaluation of Porcine circovirus (PCV) 2d Vaccine Strains to Protect against Currently Prevalent PCV2.

Porcine circovirus (PCV) 2d is a common genotype in South Korea, and the cross-protective ability of PCV2a-based vaccines has been reported recently. In this study, a PCV2d vaccine candidate was selected, and its protective efficacy against the PCV2d isolate was evaluated. From 2016 to 2020, 234 PCV2d isolates were phylogenetically analyzed using open reading frame 2 (ORF2) sequences and classified into four subgroups: PCV2d-1, PCV2d-2, PCV2d-3, and PCV2d-4. Except for PCV2d-4, which consisted of ungrouped isolates, the three subgroups showed distinct differences at amino acid positions 53 and 169 in the ORF2. The detection rates of PCV2d-1, PCV2d-2, and PCV2d-3 were 36.5, 37.4, and 3.7%, respectively, and representative isolates were selected from each subgroup (QIA244, QIA126, and QIA169, respectively). In the neutralization assay, QIA244 showed the lowest neutralization efficiency among the three PCV2a-based vaccines, whereas the virus-like particles of QIA244 (rQIA244) provided broader protection against the three genotypes than did those of QIA126 and rQIA169. To further evaluate rQIA244 in pigs, the experimental groups were divided into rQIA244-vaccine (2dVac), commercial PCV2a-vaccine (2aVac), and no-vaccination (noVac) groups. The 2dVac effectively reduced the copy number of PCV2d in blood and tissues, as well as in tissue lesions, compared to the effect of 2aVac. Collectively, 2dVac provided by QIA244 ORF2 successfully demonstrated protective efficacy against the currently prevalent PCV2d in vitro neutralization and in vivo assays.

A Field Efficacy Trial of Recombinant Porcine Circovirus Type 2d Vaccine in Three Herds.

This study aimed to evaluate the efficacy of a virus-like particle (VLP) vaccine containing the open reading frame 2 of porcine circovirus type 2d (PCV2d) in a farm environment where natural infections associated with porcine circovirus-associated disease are endemic. The vaccine trial was conducted on three farms (H, M, and Y) with a history of infections including porcine reproductive and respiratory syndrome virus (PRRSV), PCV, Mycoplasma, and E. coli. Farm H, as well as farms M and Y, experienced natural PCV2 infection between 4 and 8 weeks post-vaccination (wpv), and 8 and 12 wpv, respectively. Viremia levels of all farms were significantly (p < 0.05) lower in vaccinated piglets than the control group after natural infection. In all farms, serum immunoglobulin G levels peaked at 8 wpv in the vaccinated groups, surpassing those in the control groups. Furthermore, neutralizing antibody titers were significantly (p < 0.05) higher in the vaccinated groups than the control groups in farms H and Y (0-8 wpv). However, there were no significant differences between the vaccinated and control group in neutralizing antibody titers of farm M (0-20 wpv). In terms of body weight, vaccinated piglets from all three farms showed significantly increased average weights at 12 wpv compared to the control groups. In conclusion, our study revealed noteworthy differences in viremia and body weight gain between vaccinated and control animals on three farms. As a result, this field trial of PCV2d VLP vaccine was successful in protecting piglets from natural PCV2 infection.

Immunomodulatory effects of canine mesenchymal stem cells in an experimental atopic dermatitis model.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multi-lineage cells, suggesting their future applicability in regenerative medicine and biotechnology. The immunomodulatory properties of MSCs make them a promising replacement therapy in various fields of animal research including in canine atopic dermatitis (AD), a skin disease with 10-15% prevalence. We investigated the immunomodulatory effects of MSCs in an experimental canine AD model induced by Dermatophagoides farinae extract ointment. Canine adipose tissue-derived MSCs (cAT-MSCs) were differentiated into mesodermal cell lineages at the third passage. Alterations in immunomodulatory factors in control, AD, and MSC-treated AD groups were evaluated using flow cytometric analysis, enzyme-linked immunosorbent assay, and quantitative reverse transcription PCR. In the MSC-treated AD group, the number of eosinophils decreased, and the number of regulatory T cells (Tregs) increased compared to those in the AD group. In addition, the immunoglobulin E (IgE) and prostaglandin E2 levels were reduced in the MSC-treated AD group compared to those in the AD group. Furthermore, the filaggrin, vascular endothelial growth factor, and interleukin-5 gene expression levels were relatively higher in the MSC-treated AD group than in the AD group, however, not significantly. cAT-MSCs exerted immunomodulatory effects in an AD canine model via a rebalancing of type-1 and -2 T helper cells that correlated with increased levels of Tregs, IgE, and various cytokines.

Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection.

BACKGROUND: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. OBJECTIVES: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. METHODS: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. RESULTS: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. CONCLUSIONS: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

A novel chimeric vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) I and II elicits neutralizing antibodies against both types.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important virus within the swine industry. The virus causes respiratory disease and reproductive failure. Two species of PRRSV-I and II are co-dominant, yet no effective vaccination strategy has been developed to protect against these two types. With an aim to develop a chimeric vaccine strain to protect against both types, in this study, a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) type I and II was rescued using reverse genetics for the first time. Four chimeric infectious clones were designed based on the genomic arrangement of the structural proteins. However, only the clone carrying the transcriptional regulatory sequence (TRS) and ORF6 of a PRRSV-I and ORF6 of a PRRSV-II generated a viable recombinant virus, suggesting that concurrent expression of ORF6 from both parental viruses is essential for the recovery of type I and II chimeric PRRSV. The chimeric virus showed significantly lower replication ability than its parental strains in vitro, which was improved by serial passaging. In vivo, groups of pigs were inoculated with either the chimeric virus, one of the parental strains, or PBS. The chimeric virus replicated in pig tissue and was detected in serum 7 days post-inoculation. Serum neutralization tests indicated that pigs inoculated with the chimeric virus elicited neutralizing antibodies that inhibited infection with strains of both species and with greater coverage than the parental viruses. In conclusion, the application of this technique to construct a chimeric PRRSV holds promise for the development of a highly effective modified live vaccine candidate. This is particularly significant since there are currently no approved commercial divalent vaccines available to combat PRRSV-I and II co-infections.

First Detection of Influenza D Virus Infection in Cattle and Pigs in the Republic of Korea.

Influenza D virus (IDV) belongs to the Orthomyxoviridae family, which also include the influenza A, B and C virus genera. IDV was first detected and isolated in 2011 in the United States from pigs with respiratory illness. IDV circulates in mammals, including pigs, cattle, camelids, horses and small ruminants. Despite the broad host range, cattle are thought to be the natural reservoir of IDV. This virus plays a role as a causative agent of the bovine respiratory disease complex (BRDC). IDV has been identified in North America, Europe, Asia and Africa. However, there has been no information on the presence of IDV in the Republic of Korea (ROK). In this study, we investigated the presence of viral RNA and seroprevalence to IDV among cattle and pigs in the ROK in 2022. Viral RNA was surveyed by the collection and testing of 999 cattle and 2391 pig nasal swabs and lung tissues using a real-time RT-PCR assay. IDV seroprevalence was investigated by testing 742 cattle and 1627 pig sera using a hemagglutination inhibition (HI) assay. The viral RNA positive rate was 1.4% in cattle, but no viral RNA was detected in pigs. Phylogenetic analysis of the hemagglutinin-esterase-fusion (HEF) gene was further conducted for a selection of samples. All sequences belonged to the D/Yamagata/2019 lineage. The seropositivity rates were 54.7% in cattle and 1.4% in pigs. The geometric mean of the antibody titer (GMT) was 68.3 in cattle and 48.5 in pigs. This is the first report on the detection of viral RNA and antibodies to IDV in the ROK.

Phylogenetic Analysis of G and P Genotypes of Bovine Group A Rotavirus Strains Isolated from Diarrheic Vietnam Cows in 2017 and 2018.

This study aimed to investigate the genetic diversity of G- and P-type bovine RVAs (BoRVAs) prevalent in Vietnam. Between 2017 and 2018, the prevalence of BoRVAs detected in diarrhea samples from 8 regions was as low as 1.9% (11/582). The prevalence of the G-type was 45.5% for G6 and 18.2% for G10; however, 36.3% remain unidentified. Interestingly, all BoRVAs were investigated as P[11], and there was no diversity within this P-type. Geographically, the G6 and G10 types were not identified in any specific area; rather, they occurred in both Northern and Southern Vietnam. G6P[11] and G10P[11], which are combined G- and P-types, were identified in 71.4% and 28.6% of BoRVA-positive samples, respectively. Phylogenetic tree analysis revealed that the G6-type detected in Vietnamese cows is similar to strains derived from China, Japan, and Korea, whereas the G10 type is closely related to the Chinese strain. In addition, the P11 strain detected in Vietnamese cows is similar to the Spanish and Chinese strains. The BoRVA-positive rate was higher in cows aged less than 2 months (3.2%, 3/94) than in those aged 2 months or more (1.6%, 8/488). In summary, we detected the presence of G6P11 and G10P11 BoVRAs on Vietnamese cow farms, and found that they were more predominant in young calves than in older cows.

Rescue of a Live-Attenuated Porcine Epidemic Diarrhea Virus HSGP Strain Using a Virulent Strain and a Partially Attenuated Strain.

In South Korea in 2013, the G1-based vaccine failed to prevent an outbreak of G2b-type porcine epidemic diarrhea virus (PEDV), which is more pathogenic than the traditional G1-type strain, thereby allowing the virus to spread. In 2017 and 2018, field samples were cultured sequentially on Vero cells to isolate HS (virulent) and SGP-M1 (partially attenuated) strains, respectively, of the G2b type. The HS strain harbors a single amino acid (aa) change and two aa deletions in the N-terminal domain of S1 (55I56G57E→55K56Δ57Δ). The SGP-M1 strain harbors a seven aa deletion in the C-terminal domain of S2 (1380~1386ΔFEKVHVQ). By co-infecting various animal cells with these two strains (HS and SGP-M1), we succeeded in cloning strain HSGP, which harbors the mutations present in the two original viruses. The CPE pattern of the HSGP strain was different from that of the HS and SGP-M1 strains, with higher viral titers. Studies in piglets showed attenuated pathogenicity of the HSGP strain, with no clinical symptoms or viral shedding, and histopathologic lesions similar to those in negative controls. These findings confirm that deletion of specific sequences from the S gene attenuates the pathogenicity of PEDV. In addition, HSGP strains created by combining two different strains have the potential for use as novel attenuated live vaccine candidates.

Molecular Characterization of Porcine Reproductive and Respiratory Syndrome Virus in Korea from 2018 to 2022.

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease in the Republic of Korea. Surveillance of PRRS virus (PRRSV) types is critical to tailor control measures. This study collected 5062 serum and tissue samples between 2018 and 2022. Open reading frame 5 (ORF5) sequences suggest that subgroup A (42%) was predominant, followed by lineage 1 (21%), lineage 5 (14%), lineage Korea C (LKC) (9%), lineage Korea B (LKB) (6%), and subtype 1C (5%). Highly virulent lineages 1 (NADC30/34/MN184) and 8 were also detected. These viruses typically mutate or recombine with other viruses. ORF5 and non-structural protein 2 (NSP2) deletion patterns were less variable in the PRRSV-1. Several strains belonging to PRRSV-2 showed differences in NSP2 deletion and ORF5 sequences. Similar vaccine-like isolates to the PRRSV-1 subtype 1C and PRRSV-2 lineage 5 were also found. The virus is evolving independently in the field and has eluded vaccine protection. The current vaccine that is used in Korea offers only modest or limited heterologous protection. Ongoing surveillance to identify the current virus strain in circulation is necessary to design a vaccine. A systemic immunization program with region-specific vaccinations and stringent biosecurity measures is required to reduce PRRSV infections in the Republic of Korea.

Evaluation and Determination of a Suitable Passage Number of Codon Pair Deoptimized PRRSV-1 Vaccine Candidate in Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is major economic problem given its effects on swine health and productivity. Therefore, we evaluated the genetic stability of a codon pair de-optimized (CPD) PRRSV, E38-ORF7 CPD, as well as the master seed passage threshold that elicited an effective immune response in pigs against heterologous virus challenge. The genetic stability and immune response of every 10th passage (out of 40) of E38-ORF7 CPD was analyzed through whole genome sequencing and inoculation in 3-week-old pigs. E38-ORF7 CPD passages were limited to 20 based on the full-length mutation analysis and animal test results. After 20 passages, the virus could not induce antibodies to provide effective immunity and mutations accumulated in the gene, which differed from the CPD gene, presenting a reason for low infectivity. Conclusively, the optimal passage number of E38-ORF7 CPD is 20. As a vaccine, this may help overcome the highly diverse PRRSV infection with substantially enhanced genetic stability.

Codon Pair Deoptimization (CPD)-Attenuated PRRSV-1 Vaccination Exhibit Immunity to Virulent PRRSV Challenge in Pigs.

Commercially used porcine respiratory and reproductive syndrome (PRRS) modified live virus (MLV) vaccines provide limited protection with heterologous viruses, can revert back to a virulent form and they tend to recombine with circulating wild-type strains. Codon pair deoptimization (CPD) is an advanced method to attenuate a virus that overcomes the disadvantages of MLV vaccines and is effective in various virus vaccine models. The CPD vaccine against PRRSV-2 was successfully tested in our previous study. The co-existence of PRRSV-1 and -2 in the same herd demands protective immunity against both viruses. In this study, live attenuated PRRSV-1 was constructed by recoding 22 base pairs in the ORF7 gene of the E38 strain. The efficacy and safety of the CPD live attenuated vaccine E38-ORF7 CPD to protect against virulent PRRSV-1 were evaluated. Viral load, and respiratory and lung lesion scores were significantly reduced in animals vaccinated with E38-ORF7 CPD. Vaccinated animals were seropositive by 14 days post-vaccination with an increased level of interferon-γ secreting cells. In conclusion, the codon-pair-deoptimized vaccine was easily attenuated and displayed protective immunity against virulent heterologous PRRSV-1.

Genetic Diversity of Bovine Group A Rotavirus Strains Circulating in Korean Calves during 2014 and 2018.

The purpose of this study was to investigate annual changes in BoRVA strains by examining the VP4 and VP7 genes of rotaviruses in Korean calves. Between 2014 and 2018, 35 out of 138 samples of calf diarrhea feces collected nationwide were positive for BoRVA. Further genetic characterization of the VP7 and VP4 genes of 35 BoRVA isolates identified three different G-genotypes (G6, G8, and G10) and two different P genotypes (P[5] and P[11]). The G6 genotype was most common (94.3%) in BoRVA-positive calves, followed by the P[5] genotype (82.9%). Four genotypes comprised combinations of VP4 and VP7: 80% were G6P[5], 14.2% were G6P[11], 2.9% were G8P[5], and 2.9% were G10P[11]. Susceptibility to infection was highest in calves aged < 10 days (35%) and lowest in calves aged 30−50 days (15.4%). The data presented herein suggest that the G6P[5] genotype is the main causative agent of diarrhea in Korean calves. In addition, it is predicted that G6P[5] will continue to act as a major cause of diarrhea in Korean calves.

Genetic Diversity of Porcine Group A Rotavirus Strains from Pigs in South Korea.

Porcine group A rotavirus (PoRVA; family, Reovirideae) strains cause acute viral gastroenteritis in piglets (especially suckling and weaned pigs), resulting in significant economic losses. In this study, we analyzed the VP7 and VP4 genes of PoRVA isolated between 2014 and 2018 from domestic pigs in South Korea to investigate the prevalence of predominant circulating genotypes (G and P types). The prevalence of the PoRVA antigen in the diarrheic fecal samples was 14.1% (53/377). Further genetic characterization of the VP7 and VP4 genes of 53 PoRVA isolates identified six different G-genotypes and five different P genotypes. The G4 and G9 genotypes were the most common (each 39.6%) in PoRVA-positive pigs, followed by P[7] and P[6] (33.9% and 30.1%, respectively). Because the G5 and G9 genotype vaccines are currently mainly used in South Korea, this result provides valuable epidemiological information about the genetic characteristics of PoRVA circulating on domestic pig farms. Development of a novel PoRVA vaccine that targets the current strains circulating in South Korea may be required for more effective virus control on pig farms.

Isolation and Genetic Characterization of a Bovine Coronavirus KBR-1 Strain from Calf Feces in South Korea.

Bovine coronavirus (BCoV) causes severe diarrhea in neonatal calves, winter dysentery in adult cattle, and respiratory disease in feedlot cattle, resulting in economic losses. A total of 16/140 calf diarrheic feces samples collected in South Korea between 2017 and 2018 were positive for BCoV. Phylogenetic analysis of the complete spike and hemagglutinin/esterase genes revealed that the 16 Korean BCoV strains belonged to group GIIa along with Korean strains isolated after 2000, whereas Korean BCoV strains isolated before 2000 belonged to group GI. Mice and goats inoculated with an inactivated KBR-1 strain (isolated from this study) generated higher antibody titers (96 ± 13.49 and 73 ± 13.49, respectively) when mixed with the Montanide01 adjuvant than when mixed with the Carbopol or IMS1313 adjuvants. Viral antigens were detected in the large intestine, jejunum, and ileum of calves inoculated with inactivated KBR-1 vaccine (104.0 TCID50/mL) at 14 days of post-challenge (DPC). However, no viral antigens were detected in calves vaccinated with a higher dose of inactivated KBR-1 strain (106.0 TCID50/mL) at 14 DPC, and they had high antibody titers and stable diarrhea scores. Currently, the group GIIa is prevalent in cows in South Korea, and although further research is needed in the future, the recently isolated KBR-1 strain has potential value as a new vaccine candidate.