Genetic Characterization and Evolution of Porcine Deltacoronavirus Isolated in the Republic of Korea in 2022

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that causes diarrhea in nursing piglets. Since its first outbreak in the United States in 2014, this novel porcine coronavirus has been detected worldwide, including in Korea. However, no PDCoV case has been reported since the last report in 2016 in Korea. In June 2022, the Korean PDCoV strain KPDCoV-2201 was detected on a farm where sows and piglets had black tarry and watery diarrhea, respectively. We isolated the KPDCoV-2201 strain from the intestinal samples of piglets and sequenced the viral genome. Genetically, the full-length genome and spike gene of KPDCoV-2201 shared 96.9–99.2% and 95.8–98.8% nucleotide identity with other global PDCoV strains, respectively. Phylogenetic analysis suggested that KPDCoV-2201 belongs to G1b. Notably, the molecular evolutionary analysis indicated that KPDCoV-2201 evolved from a clade different from that of previously reported Korean PDCoV strains and is closely related to the emergent Peruvian and Taiwanese PDCoV strains. Furthermore, KPDCoV-2201 had one unique and two Taiwanese strain-like amino acid substitutions in the receptor-binding domain of the S1 region. Our findings suggest the possibility of transboundary transmission of the virus and expand our knowledge about the genetic diversity and evolution of PDCoV in Korea.


Sample Collection and RNA Extraction
In June 2022, eight fecal and one intestinal tissue samples were submitted to the Animal Disease Intervention Center of Kyungpook National University from a pig farm located in Gyeongsangbuk-do. Each sample was separately suspended in phosphate-buffered saline, homogenized, and centrifuged. The supernatant of each sample was used to extract total DNA/RNA using Patho Gene-spin™ DNA/RNA Extraction Kit (iNtRON Biotechnology, Sungnam, Korea).

Isolation and Genome Sequencing of PDCoV
Sequence analysis of the viral genome was performed on seven samples with a cycle threshold (Ct) of ≤25 among the nine PDCoV-positive samples. The spike (S) protein genes were sequenced by RT-PCR using PDCoV-specific primer sets (Table S1). We attempted to isolate PDCoV from the PDCoV-positive intestinal samples using swine testis (ST) cells (ATCC CRL-1746), as described previously [31]. We designed 11 pairs of primers for full-length genome sequencing of KPDCoV-2201 based on the conserved regions of the 127 complete genome sequences available in GenBank (Table S1). The synthesis of each cDNA fragment, PCR amplification, purification of the amplified PCR products, and determination of the sequences of the 5 -and 3 -termini of the viral genome were performed as previously described by Park et al. 2022 [32]. The sequences of each product were analyzed using the Sanger's method by a commercial company (BIONICS, Daejeon, Republic of Korea). One complete genome sequence (strain KPDCoV-2201) was deposited to GenBank (accession numbers OQ718500).

Phylogenetic and Molecular Evolutionary Analyses
To determine the genetic relationship between KPDCoV-2201 and other strains, nucleotide (nt) and amino acid (aa) sequences were aligned using MAFFT [33], which is available in Geneious Prime (https://www.geneious.com, accessed on 21 July 2022), together with 127 other full-length PDCoV genome sequences and 273 complete spike gene sequences available in GenBank. Phylogenetic trees were constructed using the RAxML method available in Geneious Prime (https://www.geneious.com, accessed on 21 July 2022) based on the general time-reversible (GTR) nucleotide substitution with a gamma distribution model [34]. These trees were subjected to bootstrap analysis with 1000 replicates to determine the percentage reliability values for each internal node of the tree [35]. All phylogenetic trees were visualized using the iTOL phylogenetic tree viewer [36]. To estimate the temporal distributions of KPDCoV-2201 and the phylogenetic relationships in South Korea, we constructed a time-scaled phylogenetic tree of G1b PDCoV strains using BEAST v.1.10.4 [37] with a GTR + gamma (GTR + γ) substitution model. Time-scaled maximum clade credibility tree was constructed using TreeAnnotator v. 1

Detection and Isolation of KPDCoV-2201
In June 2022, pigs of all ages on a farm located in Gyeongsangbuk-do exhibited the clinical symptoms of watery diarrhea. Approximately 10% of the sows presented with diarrhea symptoms, whereas 90% of the piglets showed diarrhea symptoms with a mortality rate of 40%, lower than the typical mortality rate of PEDV infection ( Figure 1). Etiological diagnostic results showed that all nine samples collected from the farm were positive for PDCoV but negative for the remaining nine viral antigens (Table 1). Subsequently, seven PDCoV-positive samples with high viral titers (Ct value of <25) were selected for the sequencing of S gene. The results of the sequencing analysis showed that all S genes from all seven samples shared 100% homology at the nucleotide level. Further, we selected the intestinal sample with the highest viral load and inoculated it into ST cells for virus isolation. After one passage of the cell culture, we observed a remarkable cytopathic effect in the infected cells, indicating that the PDCoV Korean isolate was successfully isolated in this study ( Figure S1).
Pathogens 2023, 12, x FOR PEER REVIEW 3 of 10 sequences available in GenBank. Phylogenetic trees were constructed using the RAxML method available in Geneious Prime (h ps://www.geneious.com, accessed on 21 July 2022) based on the general time-reversible (GTR) nucleotide substitution with a gamma distribution model [34]. These trees were subjected to bootstrap analysis with 1000 replicates to determine the percentage reliability values for each internal node of the tree [35]. All phylogenetic trees were visualized using the iTOL phylogenetic tree viewer [36].

Detection and Isolation of KPDCoV-2201
In June 2022, pigs of all ages on a farm located in Gyeongsangbuk-do exhibited the clinical symptoms of watery diarrhea. Approximately 10% of the sows presented with diarrhea symptoms, whereas 90% of the piglets showed diarrhea symptoms with a mortality rate of 40%, lower than the typical mortality rate of PEDV infection ( Figure 1). Etiological diagnostic results showed that all nine samples collected from the farm were positive for PDCoV but negative for the remaining nine viral antigens (Table 1). Subsequently, seven PDCoV-positive samples with high viral titers (Ct value of <25) were selected for the sequencing of S gene. The results of the sequencing analysis showed that all S genes from all seven samples shared 100% homology at the nucleotide level. Further, we selected the intestinal sample with the highest viral load and inoculated it into ST cells for virus isolation. After one passage of the cell culture, we observed a remarkable cytopathic effect in the infected cells, indicating that the PDCoV Korean isolate was successfully isolated in this study ( Figure S1).

Full-Length Genomic Characterization of KPDCoV-2201
The full-length genome sequence of KPDCoV-2201 was analyzed to determine the genetic characteristics of PDCoV, which was recently detected in Korea.  (Table 2). According to a previously reported structural analysis, the S protein PDCoV contains the S1-NTD (N-terminal domain of S1), S1-CTD (C-terminal domain of S1), CH-N (N-terminal central helix), CH-C (C-terminal central helix), FP (fusion peptide), HR-N (N-terminal heptad repeat), and HR-C (C-terminal heptad repeat), and the PDCoV receptor binding domain (RBD) is located at the C-terminal of the S1 subunit (S1-CTD) of the S protein [38]. Based on the S gene of KPDCoV-2201, the aa sequences of two PDCoV strains, which are closely related to KPDCoV-2201 according to phylogenetic analysis, were aligned and compared with those of the previously reported eight Korean PDCoV strains ( Figure S2A). The alignment results revealed that KPDCoV-2201 has 18-31 aa substitutions with Korean PDCoV strains, of which 16 aa substitutions (K96R, V277L, V326I, T387S, R342K, E/K485G, K534N, I550L, A624T, A630T, L632M, N639S, R/Q642K, A706T, K815T, and E854D) were not identical to those of all Korean PDCoV sequences, and 2 aa substitutions (I190V and D284N) were identical to those of only one strain each. KPDCoV-2201 had three aa differences from the previously reported eight Korean PDCoV strains in the S1-CTD region, but two of three were identical to those of the recently emerged PDCoV in the Taiwan strain (GenBank accession no. MZ712038) ( Figure S2B).

Phylogenetic and Molecular Evolutionary Analyses
For phylogenetic analysis, a RAxML tree was constructed using the genomic sequence of the KPDCoV-2201 strain along with previously reported 127 complete genomic sequences or 273 spike gene sequences, respectively (Figure 2). According to a previous report [15], PDCoV strains were classified into two genogroups (G)s, G1 and G2. G1 is divided into G1a (China) and G1b (United States). G2 includes PDCoV originating from Southeast Asia. Based on phylogenetic analysis of the full-length genome, the PDCoV-2201 strain was classified as G1b clustered with the US-like PDCoVs from the United States, Japan, Taiwan, Peru, Haiti, and the previously reported Korean strains (Figure 2A). Moreover, in the phylogenetic tree constructed using the S gene, KPDCoV-2201 was more closely related to PDCoV strains from Taiwan and Peru and was not clustered with other Korean PDCoV strains ( Figure 2B).
To elucidate the evolutionary characteristics of Korean PDCoV strains, times to the most recent common ancestor (tMRCA) were analyzed using the S gene of strains belonging to the G1b group.

Discussion
Recent studies have reported recombinant PDCoV strains in Southeast Asia and highly pathogenic strains in China, suggesting that PDCoV exhibits continuous antigenic changes [14,15,39]. Moreover, a case of PDCoV infection in humans was reported in Haiti [40], highlighting the risk of cross-species transmission and zoonosis. Therefore, it is necessary to conduct further research to expand insights into the PDCoV infection status and its genetic epidemiology for the global distribution of the virus.
To our knowledge, there has been no study on PDCoV in Korea for approximately the past 7 years. In 2022, KPDCoV-2201, a Korean PDCoV strain, was isolated from ST cells, and its full-length genome was sequenced in this study. Phylogenetic analysis based on the 128 full-length genome sequences showed that global PDCoV strains were classified into three distinct genogroups: G1a (China group), G1b (United States group), and G2 (Southeast Asia group) as previously reported [14,15] (Figure 2). All Korean PDCoV strains, including KPDCoV-2201 isolated in this study, were classified into the G1b genogroup, but the KPDCoV-2201 was more closely related to Taiwan PDCoV strains than to previously reported Korean PDCoV strains.
Due to research gaps, it remains unclear whether the origin of KPDCoV-2201 is the result of evolutions within Korean PDCoV strains or whether it was recently introduced as part of the Asian epidemic. Therefore, to further elucidate the evolutionary characteristics of the KPDCoV-2201 strain, tMRCA analysis was conducted using the S gene sequences of strains belonging to the G1b group. As a result, the KPDCoV-2201 was classified as Clade 2 and shared the same tMRCA with recently reported Taiwanese and Peruvian strains, which differed from previously reported Korean PDCoV strains belonging

Discussion
Recent studies have reported recombinant PDCoV strains in Southeast Asia and highly pathogenic strains in China, suggesting that PDCoV exhibits continuous antigenic changes [14,15,39]. Moreover, a case of PDCoV infection in humans was reported in Haiti [40], highlighting the risk of cross-species transmission and zoonosis. Therefore, it is necessary to conduct further research to expand insights into the PDCoV infection status and its genetic epidemiology for the global distribution of the virus.
To our knowledge, there has been no study on PDCoV in Korea for approximately the past 7 years. In 2022, KPDCoV-2201, a Korean PDCoV strain, was isolated from ST cells, and its full-length genome was sequenced in this study. Phylogenetic analysis based on the 128 full-length genome sequences showed that global PDCoV strains were classified into three distinct genogroups: G1a (China group), G1b (United States group), and G2 (Southeast Asia group) as previously reported [14,15] (Figure 2). All Korean PDCoV strains, including KPDCoV-2201 isolated in this study, were classified into the G1b genogroup, but the KPDCoV-2201 was more closely related to Taiwan PDCoV strains than to previously reported Korean PDCoV strains.
Due to research gaps, it remains unclear whether the origin of KPDCoV-2201 is the result of evolutions within Korean PDCoV strains or whether it was recently introduced as part of the Asian epidemic. Therefore, to further elucidate the evolutionary characteristics of the KPDCoV-2201 strain, tMRCA analysis was conducted using the S gene sequences of strains belonging to the G1b group. As a result, the KPDCoV-2201 was classified as Clade 2 and shared the same tMRCA with recently reported Taiwanese and Peruvian strains, which differed from previously reported Korean PDCoV strains belonging to Clade 1 (Figure 3). It is noteworthy that the occurrences of Clade 2 PDCoV strains increased compared to their A previous study reported that the titers of the recently emerged Taiwanese PDCoV strains were higher than those of the strain detected in 2015, indicating severe symptoms due to single infections with these PDCoV strains [41]. In our study, we confirmed that severe clinical symptoms were observed as a consequence of a single infection with PDCoV without coinfection by the other nine types of enteric viral pathogens in sows and piglets. These results suggested that PDCoV could potentially be the primary causative agent of severe diarrhea in some pig farms. However, diagnosing for PDCoV infection may not have been routinely conducted in diagnostic laboratories since its clinical symptoms are similar to those of other major viral pathogens causing severe enteritis, such as PEDV and TGEV. Therefore, it is essential to diagnose PDCoV in domestic pig populations exhibiting the symptoms of diarrhea, and further research is warranted on the pathogenicity of the current circulating PDCoV.

Conclusions
We isolated the KPDCoV-2201 strain from a Korean domestic pig farm where sows and piglets had severe watery diarrhea. Genetic and evolutionary analysis of KPDCoV-2201 revealed that the newly identified Korean strain is distinct from previously reported Korean strains but is closely related to non-Korean strains derived from Taiwan and Peru. Our findings suggest the possibility of transboundary transmission of the virus and expand our knowledge about the genetic diversity and evolution of PDCoV in South Korea.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pathogens12050686/s1, Table S1: Primer sets for the complete genome sequencing of PDCoV in this study; Figure S1: KPDCoV-2201 strain causes cytopathic changes in ST cells; Figure S2