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.

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.

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.

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.

Haplotype of Wild Korean Boars Infected by Classical Swine Fever Virus Subgenotype 2.1d.

Classical swine fever virus (CSFV) is one of the major pathogens that causes severe economic damage to the swine industry. Circulation of CSFV in wild boars carries the potential risk of reintroducing the virus into CSFV-free pig farms. This study carried out a genetic analysis of CSFV isolates from wild boars and analyzed the mtDNA haplotypes of the wild boars. Blood samples (n = 2140) from wild Korean boars captured in 2020 were subjected to qRT-PCR to detect CSFV, which was classified as subgenotype 2.1d based on phylogenetic analysis. CSFV had been detected in wild boars only in northern regions (Gangwon and Gyeonggi) of South Korea between 2011 and 2019. However, CSFV was identified in wild boars in the more southern regions (Chungbuk and Gyeongbuk) in 2020. Based on mitochondrial DNA analysis, all wild boars with CSFV were haplotype 01 (H01). Thus, we presume that the H01 haplotype is more susceptible to CSFV. In the future, infection of wild boars by CSFV is expected to occur intermittently every year, and we predict that most wild boars infected with CSFV will be haplotype H01.

Prevalence of Bovine Viral Diarrhea Virus Infections in Pigs on Jeju Island, South Korea, from 2009-2019 and Experimental Infection of Pigs with BVDV Strains Isolated from Cattle.

On Jeju Island, South Korea, pigs have not been vaccinated against classical swine fever (CSF) since 1999. Analysis of bovine viral diarrhea virus (BVDV) isolated from pigs on Jeju Island between 2009 and 2019 identified five BVDV-1a strains and one BVDV-1b strain. These BVDV types were shown to be the same types as BVDV strains isolated from neighboring cow farms. BVDV antibody-positive pigs (both BVDV-1 and -2) were also detected at 54 of 168 pig farms during this period. In pig infection experiments using BVDV-1a and -2a strains isolated from neighboring cow farms, BVDV-1a was detected in the blood of one of four pigs infected at both 6 and 35 days post-infection (dpi) and in the blood of two of the four pigs at 28 dpi. Pigs showed higher anti-BVDV-1 titers (5.5 ± 1.5 log2) at 35 dpi. BVDV-2a was detected in the blood of one of four pigs infected with this virus at 28 dpi only, and lower antibody titers (2.75 ± 0.75 log2) were seen in these pigs at 35 dpi. While BVDV infection is not particularly pathogenic in pigs, it is still important to monitor porcine BVDV infections due to a differential diagnosis of CSFV.

Efficacy of Needle-Less Intradermal Vaccination against Porcine Epidemic Diarrhea Virus.

To prevent diarrhea in suckling piglets infected by porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PED) vaccines are administered mainly through intramuscular (IM) or oral routes. We found that growing pigs vaccinated with an inactivated PEDV vaccine via the intradermal (ID) route had higher neutralizing antibody titers and cytokine (IFN-γ, IL-4, and IL-10) levels than non-vaccinated pigs. In addition, suckling piglets acquired lactogenic immunity from pregnant sows inoculated with an ID PED vaccine. We evaluated the efficacy of vaccination via this route, along with subsequent protection against virulent PEDV. At six days post-challenge, the survival rate of suckling piglets exposed to virulent PEDV was 70% for the ID group and 0% for the mock group (no vaccine). At necropsy, villi length in the duodenum and ileum of piglets with lactogenic immunity provided by ID-vaccinated sows proved to be significant (p < 0.05) when compared with those in piglets from mock group sows. Thus, vaccination using an inactivated PED vaccine via the ID route provides partial protection against infection by virulent PEDV.

Phylogenetic analysis of kobuviruses and astroviruses from Korean wild boars: 2016-2018.

Between 2016 and 2018, the prevalence of porcine kobuvirus (PKoV) and porcine astrovirus (PAstV) in Korean wild boars (n = 845) was 28.0% and 10.7%, respectively. Coinfection by both viruses was detected in 5.1% of boars. Phylogenetic analysis revealed that 134 PKoV isolates belonged to diverse lineages within the species Aichivirus C; however, one strain (WKoV16CN-8627) clustered with bovine kobuvirus (Aichivirus B). Forty-seven PAstVs belonged to lineage PAstV4, and only one strain (WAst17JN-10931) was a novel addition to lineage PAstV2. The two viruses were more prevalent in boars weighing ≤ 60 kg than in boars weighing > 61 kg.

Genetic analysis of porcine parvoviruses detected in South Korean wild boars.

Porcine parvovirus 1 (PPV1) is a major cause of reproductive failure in pigs. To date, six additional porcine parvoviruses (PPV2-PPV7) have been identified. In this study, we detected 11 PPV1 strains, five PPV3 strains, three PPV4 strains, six PPV5 strains, five PPV6 strains, and one PPV7 strain in Korean wild boars. PPV1, -3, and -5, and PPV6 from Korean wild boars harbor conserved motifs within the Ca2+ binding loop and the catalytic center of the PLA1 motif. Intra-species recombination among PPV7 strains was also identified. Genetic characterization revealed that PPV1 from Korean wild boars may be similar to virulent PPV strains.

Comparative Analysis of the Productivity and Immunogenicity of an Attenuated Classical Swine Fever Vaccine (LOM) and an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns) from Laboratory to Pig Farm.

Herein, we compared the productivity of pigs inoculated with one of two classical swine fever (CSF) vaccines (low virulent of Miyagi (LOM) or Flc-LOM-BErns) plus the swine erysipelothrix rhusiopathiae (SE) vaccine. The feed intake and weight increase of the pigs inoculated with Flc-LOM-BErns + SE were normal. However, the feed intake of the pigs inoculated with LOM + SE dropped sharply from four days post-vaccination (dpv). In addition, the slaughter date was an average of eight days later than that of the pigs inoculated with Flc-LOM-BErns + SE. All pigs inoculated with the Flc-LOM-BErns + SE vaccine were completely differentiated at 14 days against CSF Erns antibody and at approximately 45 days against the bovine viral diarrhea virus (BVDV) Erns antibody; the titers were maintained until slaughter. Leucopenia occurred temporarily in the LOM + SE group, but not in the Flc-LOM-BErns + SE group. Expression of tumor necrosis factor (TNF)-α and IFN-γ was significantly (p < 0.05) higher in the LOM + SE group than in the mock (no vaccine) group. When conducting the same experiment on a breeding farm, the results were similar to those of the laboratory experiments. In conclusion, the biggest advantage of replacing the CSF LOM vaccine with the Flc-LOM-BErns vaccine is improved productivity.

Complete Genome Sequences of Two Type 3 Porcine Circoviruses, WB17KW and WB20GG, Isolated from Korean Wild Boar.

Porcine circovirus type 3 (PCV3) in domestic pigs was first reported in South Korea in 2017. Here, we report the first complete genome sequences of two PCV3 strains isolated from Korean wild boar, which enhance our understanding about the genetic relatedness of PCV3 in domestic pigs and wild boar.

Protection of Piglets with Maternally Derived Antibodies from Sows Inoculated with an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns).

Here, we investigated the protective efficacy provided by passive immunity induced by a classical swine fever (Flc-LOM-BErns) vaccine with the newly developed DIVA (Differentiating Infected from Vaccinated Animals) function. Ten pigs (aged 40-60 days) with maternally derived antibodies (MDAs) obtained from sows inoculated with the Flc-LOM-BErns vaccine were challenged with virulent classical swine fever virus (CSFV). Pigs with an MDA titer of 6 log2 induced by the Flc-LOM-BErns vaccine were fully protected against virulent CSFV challenge but not the pigs with an MDA titer under 5 log2. In addition, Flc-LOM-BErns vaccine-derived MDAs successfully differentiated vaccinated pigs by bovine viral diarrhea virus (BVDV) Erns/CSFV Erns antibody detection, functioning as a DIVA.

Prevalence and Genetic Diversity of Atypical Porcine Pestivirus (APPV) Detected in South Korean Wild Boars.

Atypical porcine pestivirus (APPV), currently classified as pestivirus K, causes congenital tremor (CT) type A-II in piglets. Eighteen APPV strains were identified from 2297 South Korean wild boars captured in 2019. Phylogenetic analysis of the structural protein E2 and nonstructural proteins NS3 and Npro classified the APPV viruses, including reference strains, into Clades I, II and III. Clade I was divided into four subclades; however, the strains belonging to the four subclades differed slightly, depending on the tree analysis, the NS3, E2, and Npro genes. The maximum-likelihood method was assigned to South Korean wild boar APPV strains to various subclades within the three trees: subclades I.1 and I.2 in the E2 tree, subclade I.1 in the Npro tree, and subclades I.1 and I.4 in the NS3 ML tree. In conclusion, APPV among South Korean wild boars belonging to Clade I may be circulating at a higher level than among the South Korean domestic pig populations.

Genetic Diversity of Porcine Circovirus Isolated from Korean Wild Boars.

In Korea, three genotypes of porcine circovirus type 2 (PCV2a, PCV2b, and PCV2d) have been identified on domestic pig farms, while two genotypes (PCV2a and PCV2b) have been identified in wild boar populations. Here, we investigated genotype diversity and genotypic shift in 91 PCV2 isolates from 1340 wild boars captured in South Korea between 2013 and 2017. Phylogenetic analyses based on the complete ORF2 showed that the 91 PCV2 strains were detected as four genotypes by qPCR screening assay: PCV2a (2.2%, 2/91), PCV2b (16.5%, 15/91), PCV2d (80.2%, 73/91), and PCV2h (1.1%, 1/91). Only one intergenotype recombinant event was detected between PCV2 ORF2 in wild boars (PCV2b) and domestic pigs (PCV2a). Amino acid positions 86-89 within ORF2, which distinguishes the different genotypes, were conserved in all PCV2 genotypes isolated from South Korean wild boars, including TNKI in PCV2a/PCV2h, SNPR in PCV2b, and SNPL in PCV2d. The estimated nucleotide substitution rates in the ORF2 region of viruses from South Korean wild boars and domestic pigs were 5.8145 × 10-4 and 4.5838 × 10-4 substitutions per site per year (s/s/y), respectively. The times to the most recent common ancestor (tMRCA) for South Korean domestic pig PCV2 were 1937 (PCV2a), 1972 (PCV2b), 1999 (PCV2d-1), and 2000 (PCV2d-2). By contrast, the tMRCA for South Korean wild boar PCV2b and PCV2d were 1989 and 2001, respectively. Thus, the PCV2d genotype is prevalent among South Korean wild boars and domestic pigs.