{"paper_id":"47516f4e-302c-4af3-b7f1-e1519e8274dd","body_text":"Genotypic characterization of novel S-DEL variants of porcine epidemic diarrhea virus identified in South Korea | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genotypic characterization of novel S-DEL variants of porcine epidemic diarrhea virus identified in South Korea Duri Lee, Sungrae Kim, Yunhee Gim, Changhee Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4053243/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The highly pathogenic-genotype 2b (HP-G2b) porcine epidemic diarrhea virus (PEDV) that caused the 2013–2014 pandemic has evolved in South Korea and has endemically affected the domestic pig industry. This study describes the genotypic traits of novel HP-G2b PEDV strains identified in affected farms experiencing low disease severity with < 10% neonatal mortality. Nucleotide sequencing revealed common deletion (DEL) patterns of duad residues, termed S-DEL2, at positions 60 and 61, 61 and 62, or 63 and 64 in the N-terminal domain of the spike (S) gene of all isolates. Nevertheless, the S barcode profiles of S-DEL2 variants differed from each other and shared 96.0–99.4% and 98.5–99.6% homology with other South Korean HP-G2b PEDV strains at the S and complete genome levels, respectively. Moreover, genetic and phylogenetic analyses confirmed that the S-DEL2 strains belonged to diverse domestic clades, CK, CK.1, CK.2, or NC. The emergence of novel S-DEL2 strains suggests continuous evolution of PEDV under endemic circumstances, which may result in genetic diversity and distinct clinical presentations. Overall, this study advances our knowledge regarding the genetic and pathogenic heterogeneity of PEDV and emphasizes the significance of active monitoring and surveillance to identify novel variants and further explore their genotypic and phenotypic characteristics. Figures Figure 1 Figure 2 Full Text Porcine epidemic diarrhea virus (PEDV) is a fatal swine enteric coronavirus that belongs to the subgenus Pedecovirus of the genus Alphacoronavirus within the family Coronaviridae of the order Nidovirales [1, 2]. PEDV is a large, enveloped virus with a non-segmented, positive-sense RNA genome of approximately 28 kb comprising seven canonical coronaviral genes [1, 3]. The virus can be classified into two major genotypes with at least two sub-genotypes: G1 (classical G1a and recombinant G1b) and G2 (local epidemic G2a and global epidemic G2b) [1, 3]. The advent of the highly pathogenic (HP)-G2b PEDV resulted in a pandemic that ruined pig-producing nations in America and Asia during 2013–2014, posing socioeconomic threats to the global swine industry [1, 3]. South Korea also suffered from explosive nationwide outbreaks of HP-G2b PEDV, causing the loss of an estimated one million neonatal piglets during 2013–2014 [3, 4]. Since then, HP-G2b PEDV has continued to cause nationwide epidemics each year and has evolved along with genetic divergence [1]. Consequently, the HP-G2b PEDV strains circulating in South Korea are genetically clustered into eight clades with two subclades on the basis of geographical origin [1]. From December 2023 to January 2024, acute outbreaks of severe diarrheic diseases accompanied by low neonatal mortality (<10%) were reported in multiple unvaccinated farrow-to-finish herds in southern South Korea. Clinical samples (feces or small intestine) collected from diarrheic or dead piglets at seven farms were submitted to our laboratory for diagnosis and subjected to virus-specific RT-PCR assays as described elsewhere [5–7]. All specimens from the seven farms were PEDV-positive, with cycle threshold (Ct) values ranging from 14.4 to 27.2; however, no other viral pathogens causing neonatal diarrhea were identified in these cases. We then determined the entire sequences of the spike (S) genes of the seven isolates detected from each farm as previously published [5–7] and deposited their sequence data in the GenBank database under accession numbers PP441968–441974. Nucleotide (nt) sequencing analysis revealed genetic divergence (different barcode profiles representing a distinct locus from the HP-G2b prototype sequence) among the strains designated as GNU-2389–92 and GNU-2401–3 (Fig. 1), exhibiting 96.4%–98.8% amino acid (aa) homology, and displayed 96.0%–99.4% aa identity to the South Korean HP-G2b strains (Supplementary Table S1). All strains belonged to HP-G2b with the genetic marker, S insertions and deletions (INDELs), compared with the classical G1a prototype CV777 strain [1, 3, 7]. Interestingly, unique deletion (DEL) signatures were identified in the N-terminal domain (NTD; residues 19–233) of S1 from all isolates in comparison with the HP-G2b South Korean prototype strain KNU-141112. The S genes of all PEDV strains determined in this study harbored a novel deletion (DEL) of duad (GV), (VN), or (ST) aa residues at positions 60 and 61 (GNU-2389), 61 and 62 (GNU-2390–2 and GNU-2401–2), or 63 and 64 (GNU-2403), respectively, in their S1 NTD, named S-DEL2 (Fig. 1A). These S-DEL2 patterns in S1 NTD were unprecedented in the genome sequence of other PEDV field strains retrieved from the GenBank database. Due to the DEL of duad ( 60 GV 61 ), ( 61 VN 62 ), or ( 63 ST 64 ) residues in the S1 NTD of the S-DEL2 variants, the size of their S genes was 4,155-nt long, encoding a 1,384-aa protein, which was 6-nt shorter than those of the conventional HP-G2b strains. Further prediction of N-linked glycosylation sites confirmed the loss of one putative N-glycosylation sequon ‘NST’ at N62 on the S1 NTD of all isolates, except for GNU-2389, which is commonly found in classical HP-G2b field viruses (Fig. 1A). To further uncover the genotypic features of the S-DEL2 variants, their complete genomic sequences were deciphered using the Sanger method as described previously [5–7]. We could not achieve whole genome sequencing (WGS) of four isolates directly from the clinical cases, probably because of high Ct values (>25), representing the low viral load present in the respective PEDV-positive samples. However, the remaining three isolates, GNU-2389, -2391, and -2401, whose full-length genomes were successfully sequenced, and the genomic sequences were deposited at GenBank under accession numbers PP441972–441974. WGS revealed no additional INDELs throughout the entire genome of the fully sequenced S-DEL2 strains, except for the S-coding region (Fig. 1B). The S-DEL2 viruses shared a high level of nt sequence similarity with each other (98.7%–98.8%) and with other domestic HP-G2b strains at the genome level (98.5%–99.6%) (Supplementary Table S2). Detailed information, including the number of nt or aa differences and the percentage of homology between each S-DEL2 variant and the domestic prototype strain KNU-141112, is shown in Supplementary Table S3. To verify genetic relatedness, we conducted phylogenetic analyses using the S and full-length genome of PEDV determined in this study and those accessible from GenBank, as described previously (Fig. 2) [5–7]. As depicted in Fig. 2A, the S protein-based phylogeny clearly demonstrated four genotypes, viz., G1a, G1b, G2a, and G2b. The South Korean G2b strains were further categorized into six clades (NW, KJ, CK, JH, JD, and NC) and two subclades (CK.1 and CK.2). The novel S-DEL2 variants belonged to the G2b cluster along with the global and domestic HP-G2b strains and were classified into CK (GNU-2402), CK.1 (GNU-2403), CK.2 (GNU-2389), or NC (GNU-2390–2 and GNU-2401). Similarly, whole-genome phylogeny indicated that the S-DEL2 strains were clustered within the same genogroup as the global and domestic G2b strains (Fig. 2B). Since the national catastrophe of HP-G2b PEDV during 2013–2014, this virus has become endemic in South Korea and has evolved to undergo genetic and pathogenic heterogeneity [1]. Recently, PEDV outbreaks with reduced morbidity and mortality occurred in several commercial swine farms with no previous record of PEDV infection or vaccination. Although we initially considered the concurrent introduction of the genetically identical PEDV strain into those farms, our genetic and phylogenetic analyses revealed distinct differences in the barcode profile and clade categories among the HP-G2b viruses identified in the affected herds. Despite the abovementioned genetic varieties, these viruses demonstrated genetic similarity with the novel S-DEL2 signature at positions 60 and 61, 61 and 62, or 63 and 64 in the S1 NTD. The PEDV S protein mediates viral entry into target cells and thus triggers host immunity to produce neutralizing antibodies [3]. In particular, the S1 NTD contains one neutralizing epitope region, termed NTD/S0 (residues 19–220) (Fig. 1A) [8]. Moreover, various novel G2b variants with small or large INDELs in S1 have been reported in multiple countries, suggesting their association with virulence [9]. Therefore, genetic drift, including INDELs, and shift (i.e., recombination) in the S gene can allow PEDV to evade the host antibody response and/or alter viral pathogenicity [5, 9–11]. The S-DEL2 viruses lost the N62 glycan motif on the NTD/S0 domain of S, resulting from the emergence of S-DEL2 in the S1 NTD, possibly causing the modification of the S protein conformation that may contribute to the antigenic variation of PEDV. In fact, the S-DEL2 variants exhibited a conformational change on residues 51–69 residing on the NTD/S0 region compared with the S 3D structure of KNU-141112, which in turn may confer an immune evasion strategy to the virus (Supplementary Fig. S1). Considering the disease severity in S-DEL2 virus-infected farms, the DEL of duad residues in the S1 NTD may be associated with PEDV virulence in neonatal piglets. The S1 NTD is responsible for sialic acid (SA) binding, which facilitates viral entry by recruiting host SA-containing components to invade the thick mucus barrier close to the enterocytes [12]. More interestingly, the pentad “GENQG” at positions 56–60 in the S1 NTD was proven to be the major residue for the SA binding activity of the PEDV S protein, and infectious clone-derived mutants with the duad (GE) or pentad (GENQG) DEL displayed the attenuation of SA-dependency and virulence [11]. Because the duad “ 60 GV 61 ”, “ 61 VN 62 ”, or “ 63 ST 64 ” residues are located next to or adjacent to the pentad “ 56 GENQG 60 ” in the S1 NTD, it is anticipated that the S-DEL2 variants would weaken the SA-dependent viral entry and diminish the pathogenicity of PEDV. Considering the absence of such field S-DEL2 isolates that can grow in cell culture, the cutting-edge application of reverse genetics (RG) is essential for pursuing loss-of-function research to corroborate the abovementioned aspects. With the availability of the PEDV RG platform in our laboratory [10], our next step is to investigate the precise role of S-DEL2 in the antigenicity, SA dependency, and virulence of PEDV. Nevertheless, our sequence data provide insights into understanding the genetic and pathogenic variation of PEDV field strains. The present study also underscores the need for implementing continuous monitoring and surveillance investigations, followed by swift data sharing and accessibility, to establish PEDV management for preventing future outbreaks/pandemic. Declarations Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00251145). Ethical declarations Conflict of interest : The authors declare that they have no conflict of interest. Ethical approval : This article does not contain any studies with animals performed by any of the authors. References Jang G, Lee D, Shin S, Lim J, Won H, Eo Y, Kim CH, Lee C (2023) Porcine epidemic diarrhea virus: an update overview of virus epidemiology, vaccines, and control strategies in South Korea. J Vet Sci 24:e58 Schoch CL, Ciufo S, Domrachev M, Hotton CL, Kannan S, Khovanskaya R, Leipe D, Mcveigh R, O'Neill K, Robbertse B, Sharma S, Soussov V, Sullivan JP, Sun L, Turner S, Karsch-Mizrachi I (2020) NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database 2020:baaa062 Lee C (2015) Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virol J 12:193 Lee S, Lee C (2014) Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, 2013. Emerg Infect Dis 20:1223–1226 Park J, Lee C (2020) Emergence and evolution of novel G2b-like porcine epidemic diarrhea virus inter-subgroup G1b recombinants. Arch Virol 165:2471–2478 Jang G, Park J, Lee C (2021) Successful eradication of porcine epidemic diarrhea in an enzootically infected farm: a two-year follow-up study. Pathogens 10:830 Lee S, Lee DU, Noh YH, Lee SC, Choi HW, Yang HS, Seol JH, Mun SH, Kang WM, Yoo HS, Lee C (2019) Molecular characteristics and pathogenic assessment of porcine epidemic diarrhea virus isolates from the 2018 endemic outbreaks on Jeju Island, South Korea. Transbound Emerg Dis 66:1894–1909 Li C, Li W, Lucio de Esesarte E, Guo H, van den Elzen P, Aarts E, van den Born E, Rottier PJM, Bosch BJ (2017) Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies. J Virol 91:e00273–e00217 Jang G, Park J, Lee C (2019) Complete genome sequences of novel S-deletion variants of porcine epidemic diarrhea virus identified from a recurrent outbreak on Jeju Island, South Korea. Arch Virol 164:2621–2625 Jang G, Lee D, Lee C (2022) Development of a next-generation vaccine platform for porcine epidemic diarrhea virus using a reverse genetics system. Viruses 14:2319 Jang G, Min KC, Lee IH, Won H, Yoon IJ, Kang SC, Lee C (2023) Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets. Vet microbiol 280:109727 Li W, van Kuppeveld FJM, He Q, Rottier PJM, Bosch BJ (2016) Cellular entry of the porcine epidemic diarrhea virus. Virus Res 226:117–127 Supplementary Files SupplementaryfileGenBankentries.pdf Supplementaryfile.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 30 Mar, 2024 Reviewers invited by journal 27 Mar, 2024 Editor assigned by journal 13 Mar, 2024 First submitted to journal 11 Mar, 2024 Editorial decision: Minor Revision 11 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4053243\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":284602173,\"identity\":\"98a4e400-f0cb-4985-a7e6-4cca9ebd0a49\",\"order_by\":0,\"name\":\"Duri Lee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Gyeongsang National University College of Veterinary Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Duri\",\"middleName\":\"\",\"lastName\":\"Lee\",\"suffix\":\"\"},{\"id\":284602174,\"identity\":\"fa611bbe-8351-4cd2-88de-e733a386a971\",\"order_by\":1,\"name\":\"Sungrae Kim\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Gyeongsang National University College of Veterinary Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sungrae\",\"middleName\":\"\",\"lastName\":\"Kim\",\"suffix\":\"\"},{\"id\":284602175,\"identity\":\"fe033308-ffab-40ff-89d1-695d60f5400e\",\"order_by\":2,\"name\":\"Yunhee Gim\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Gyeongsang National University College of Veterinary Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yunhee\",\"middleName\":\"\",\"lastName\":\"Gim\",\"suffix\":\"\"},{\"id\":284602176,\"identity\":\"49956e9c-9389-4d8a-b48f-282a908b4804\",\"order_by\":3,\"name\":\"Changhee Lee\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACCSBmbGBg4EcWIE6LZAPJWgwOEKuFf3bzs4dfd9jIGZ9fe0yCocaOQXL2AfxaJO4cMzeWPZNmbHbjXZoEw7FkBmm+BPxaDCQSzKQl2w4nbrtxxkyCge0AgxwPAYcZSKR/A2r5X795BkjLP6K05JhJfmw7kGDA32Mmwdh2gEGakBaJGzll0oxtyYYzbvAYWyT2JfNI9hDQwj8jfZvkzzY7ef7+M4Y3Pnyzk5M4Q0ALCDCDnSKRwMAARIScBQGMP8D2HSBK8SgYBaNgFIxAAACpsDzSGq/gvwAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0002-5930-5461\",\"institution\":\"Gyeongsang National University College of Veterinary Medicine\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Changhee\",\"middleName\":\"\",\"lastName\":\"Lee\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-03-09 07:57:58\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4053243/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4053243/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":53874628,\"identity\":\"617ae824-5934-47ec-ba34-6affda0ae422\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:21:50\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":128881,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSchematic diagram of genetic variation in the S gene and whole genome between the prototype strain KNU-141112 and each PEDV S-DEL2 strain. (A) The top illustration represents the organization of the S protein consisting of S1 and S2 subunits that contain a signal peptide (SP), a fusion peptide (FP), heptad repeat regions (HR1 and HR2), and a transmembrane domain (TM). Orange areas in the illustration depicting S represent four neutralizing epitopes (NTD/S0, residues 19–220; COE, residues 502–641; residues 744–774; residues 1371–1377) of HP-G2b PEDV. A schematic diagram (barcode profiles) of the S gene alignment in relation to the KNU-141112 S sequence was generated using the Geneious software version 2023.2.1. Lightly shaded regions are those identical to KNU-141112 (thick horizontal black line), and each vertical black bar represents one amino acid sequence differently from KNU-141112. Thin horizontal dashed lines indicate deleted amino acids. The digits in parentheses on the right indicate the number of amino acid changes and the percentage identity compared with KNU-141112. Potential N-glycosylation amino acid sites (NXS/T, where X ≠ P) are shown as red (inverted) triangles or inverted triangles. The loss (N62 and N381) and gain (N128, N724, and N1194) of glycosylation sequons are indicated by blue and purple inverted triangles and green triangles, respectively. The amino acid sequence alignment of the partial S1-NTD domain (residues 1–100) between KNU-141112 and S-DEL2 strains is presented at the bottom. The deletion of duad residues at positions 60-64, including an N-glycan sequon at N62, is highlighted in yellow. (B) The top diagram represents the complete genome organization of PEDV, with bars symbolizing the 5′ UTR-ORF1a-ORF1b-S-ORF3-E-M-N-3′ UTR and brown arrows indicating 16 nonstructural proteins (nsp1–16). A schematic diagram (barcode profiles) of the whole genome alignment in relation to the KNU-141112 genome sequence is presented at the bottom. Lightly shaded regions are those identical to KNU-141112 (thick horizontal black line), and each vertical black bar represents one amino acid sequence differently from KNU-141112. Thin horizontal dashed lines indicate the deleted amino acids in S.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFigure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4053243/v1/0b23311275a7f9ed7390fe58.png\"},{\"id\":53875125,\"identity\":\"b49b77b0-3791-4491-a5b9-19ba719494e4\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:29:50\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":122182,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhylogenetic analyses\\u003cstrong\\u003e \\u003c/strong\\u003ebased on\\u003cstrong\\u003e \\u003c/strong\\u003ethe S (A) and whole-genome (B) sequences of the PEDV strains. (A) Four S gene-based genotypes, G1a (red), G1b (blue), G2a (green), and G2b (navy), are indicated. Different colored strips on the navy (G2b) branches indicate the HP-G2b PEDV strains identified nationwide in South Korea that clustered into eight clades with two subclades: NW (orange), KJ (dark red), CK (light green), CK.1 (red), CK.2 (blue), JH (sky blue), JD (neon), and not classified NC (purple) clades. Different colored dots indicate the S-DEL2 strains identified in this study (light green, red, blue, and purple) and the HP-G2b Korean prototype strain KNU-141112 (black). (B) Whole-genome phylogeny indicates two genogroups, G1 (red) and G2 (navy). Scale bars indicate nucleotide substitutions per site.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFigure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4053243/v1/4129a043b08f3247b3862f52.png\"},{\"id\":53875953,\"identity\":\"ff5f25e9-75ac-44c7-a1c4-756c383f86e1\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:37:50\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":435859,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4053243/v1/e0c50c8d-eea8-4aad-8661-f54e2324670c.pdf\"},{\"id\":53874631,\"identity\":\"ec7d2d39-6b40-4ff2-b62e-a9d36e614988\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:21:50\",\"extension\":\"pdf\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":363596,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryfileGenBankentries.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4053243/v1/73e9d7db7fed3d5eee07c21b.pdf\"},{\"id\":53874630,\"identity\":\"433d3ccc-35a0-4fa9-b148-39c59ca4ec9f\",\"added_by\":\"auto\",\"created_at\":\"2024-04-01 16:21:50\",\"extension\":\"pdf\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1477615,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementaryfile.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4053243/v1/7c07fadf2311ccb0b8df11f3.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Genotypic characterization of novel S-DEL variants of porcine epidemic diarrhea virus identified in South Korea\",\"fulltext\":[{\"header\":\"Full Text\",\"content\":\"\\u003cp\\u003ePorcine epidemic diarrhea virus (PEDV) is a fatal swine enteric coronavirus that belongs to\\u0026nbsp;the subgenus \\u003cem\\u003ePedecovirus\\u003c/em\\u003e of the genus \\u003cem\\u003eAlphacoronavirus\\u003c/em\\u003e within the family \\u003cem\\u003eCoronaviridae\\u003c/em\\u003e of the order \\u003cem\\u003eNidovirales\\u003c/em\\u003e [1, 2].\\u0026nbsp;PEDV is a large, enveloped virus with\\u0026nbsp;a non-segmented, positive-sense\\u0026nbsp;RNA genome of\\u0026nbsp;approximately 28 kb comprising seven canonical coronaviral genes [1, 3].\\u0026nbsp;The virus can be classified into two major genotypes with at least two sub-genotypes: G1 (classical G1a and recombinant G1b) and G2 (local epidemic G2a and global epidemic G2b) [1, 3]. The advent of the highly pathogenic (HP)-G2b PEDV resulted in a pandemic that ruined pig-producing nations in America and Asia during 2013\\u0026ndash;2014, posing socioeconomic threats to the global swine industry [1, 3]. South Korea also suffered from explosive nationwide outbreaks of HP-G2b PEDV, causing the loss of an estimated one million neonatal piglets during 2013\\u0026ndash;2014 [3, 4]. Since then, HP-G2b PEDV has continued to cause nationwide epidemics each year and has evolved along with genetic divergence [1]. Consequently, the HP-G2b PEDV strains circulating in South Korea are genetically clustered into eight clades with two subclades on the basis of geographical origin [1].\\u003c/p\\u003e\\n\\u003cp\\u003eFrom December 2023 to January 2024, acute outbreaks of severe diarrheic diseases accompanied by low neonatal mortality (\\u0026lt;10%) were reported in multiple unvaccinated farrow-to-finish herds in southern South Korea. Clinical samples (feces or small intestine) collected from diarrheic or dead piglets at seven farms were submitted to our laboratory for diagnosis and subjected to virus-specific RT-PCR assays as described elsewhere [5\\u0026ndash;7]. All specimens from the seven farms were PEDV-positive, with cycle threshold (Ct) values ranging from 14.4 to 27.2; however, no other viral pathogens causing neonatal diarrhea were identified in these cases. We then determined the entire sequences of the spike (S) genes of the seven isolates detected from each farm as previously published [5\\u0026ndash;7] and deposited their sequence data in the GenBank database under accession numbers PP441968\\u0026ndash;441974. Nucleotide (nt) sequencing analysis revealed genetic divergence (different barcode profiles representing a distinct locus from the HP-G2b prototype sequence) among the strains designated as GNU-2389\\u0026ndash;92 and GNU-2401\\u0026ndash;3 (Fig. 1), exhibiting 96.4%\\u0026ndash;98.8% amino acid (aa) homology, and displayed 96.0%\\u0026ndash;99.4% aa identity to the South Korean HP-G2b strains (Supplementary Table S1). All strains belonged to HP-G2b with the genetic marker, S insertions and deletions (INDELs), compared with the classical G1a prototype CV777 strain [1, 3, 7]. Interestingly, unique deletion (DEL) signatures were identified in the N-terminal domain (NTD; residues 19\\u0026ndash;233) of S1 from all isolates in comparison with the HP-G2b South Korean prototype strain KNU-141112. The S genes of all PEDV strains determined in this study harbored a novel deletion (DEL) of duad (GV), (VN), or (ST) aa residues at positions 60 and 61 (GNU-2389), 61 and 62 (GNU-2390\\u0026ndash;2 and GNU-2401\\u0026ndash;2), or 63 and 64 (GNU-2403), respectively, in their S1 NTD, named S-DEL2 (Fig. 1A). These S-DEL2 patterns in S1 NTD were unprecedented in the genome sequence of other PEDV field strains retrieved from the GenBank database. Due to the DEL of duad (\\u003csup\\u003e60\\u003c/sup\\u003eGV\\u003csup\\u003e61\\u003c/sup\\u003e), (\\u003csup\\u003e61\\u003c/sup\\u003eVN\\u003csup\\u003e62\\u003c/sup\\u003e), or (\\u003csup\\u003e63\\u003c/sup\\u003eST\\u003csup\\u003e64\\u003c/sup\\u003e) residues in the S1 NTD of the S-DEL2 variants, the size of their S genes was 4,155-nt long, encoding a 1,384-aa protein, which was 6-nt shorter than those of the conventional HP-G2b strains. Further prediction of N-linked glycosylation sites confirmed the loss of one putative N-glycosylation sequon \\u0026lsquo;NST\\u0026rsquo; at N62 on the S1 NTD of all isolates, except for GNU-2389, which is commonly found in classical HP-G2b field viruses (Fig. 1A).\\u003c/p\\u003e\\n\\u003cp\\u003eTo further uncover the genotypic features of the S-DEL2 variants, their complete genomic sequences were deciphered using the Sanger method as described previously [5\\u0026ndash;7]. We could not achieve whole genome sequencing (WGS) of four isolates directly from the clinical cases, probably because of high Ct values (\\u0026gt;25), representing the low viral load present in the respective PEDV-positive samples. However, the remaining three isolates, GNU-2389, -2391, and -2401, whose full-length genomes were successfully sequenced, and the genomic sequences were deposited at GenBank under accession numbers PP441972\\u0026ndash;441974. WGS revealed no additional INDELs throughout the entire genome of the fully sequenced S-DEL2 strains, except for the S-coding region (Fig. 1B). The S-DEL2 viruses shared a high level of nt sequence similarity with each other (98.7%\\u0026ndash;98.8%) and with other domestic HP-G2b strains at the genome level (98.5%\\u0026ndash;99.6%) (Supplementary Table S2). Detailed information, including the number of nt or aa differences and the percentage of homology between each S-DEL2 variant and the domestic prototype strain KNU-141112, is shown in Supplementary Table S3.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTo verify genetic relatedness, we conducted phylogenetic analyses using the S and full-length genome of PEDV determined in this study and those accessible from GenBank, as described previously (Fig. 2)\\u0026nbsp;[5\\u0026ndash;7]. As depicted in Fig. 2A, the S protein-based phylogeny clearly demonstrated four genotypes, viz., G1a, G1b, G2a, and G2b. The South Korean G2b strains were further categorized into six clades (NW, KJ, CK, JH, JD, and NC) and two subclades (CK.1 and CK.2). The novel S-DEL2 variants belonged to the G2b cluster along with the global and domestic HP-G2b strains and were classified into CK (GNU-2402), CK.1 (GNU-2403), CK.2 (GNU-2389), or NC (GNU-2390\\u0026ndash;2 and GNU-2401). Similarly, whole-genome phylogeny\\u0026nbsp;indicated that the S-DEL2 strains were clustered within the same genogroup as the global and domestic G2b strains (Fig. 2B).\\u003c/p\\u003e\\n\\u003cp\\u003eSince the national catastrophe of HP-G2b PEDV during\\u0026nbsp;2013\\u0026ndash;2014,\\u0026nbsp;this virus has become endemic in South Korea and has evolved to undergo genetic and pathogenic heterogeneity [1]. Recently, PEDV outbreaks with reduced morbidity and mortality occurred in several commercial swine farms with no previous record of PEDV infection or vaccination.\\u0026nbsp;Although we initially considered the concurrent introduction of the genetically identical PEDV strain into those farms, our genetic and phylogenetic analyses revealed distinct differences in the barcode profile and clade categories among the HP-G2b viruses identified in the affected herds.\\u0026nbsp;Despite the abovementioned genetic varieties, these viruses demonstrated genetic similarity with the novel S-DEL2 signature at positions 60 and 61, 61 and 62, or 63 and 64 in the S1 NTD. The PEDV S protein mediates viral entry into target cells and thus triggers host immunity to produce neutralizing antibodies [3]. In particular, the S1 NTD contains one neutralizing epitope region, termed NTD/S0 (residues 19\\u0026ndash;220) (Fig. 1A) [8]. Moreover, various novel G2b variants with small or large INDELs in S1 have been reported in multiple countries, suggesting their association with virulence [9]. Therefore, genetic drift, including INDELs, and shift (i.e., recombination) in the S gene can allow PEDV to evade the host antibody response and/or alter viral pathogenicity [5, 9\\u0026ndash;11]. The S-DEL2 viruses lost the N62 glycan motif on the NTD/S0 domain of S, resulting from the emergence of S-DEL2 in the S1 NTD, possibly causing the modification of the S protein conformation that may contribute to the antigenic variation of PEDV. In fact, the S-DEL2 variants exhibited a conformational change on residues 51\\u0026ndash;69 residing on the NTD/S0 region compared with the S 3D structure of KNU-141112, which in turn may confer an immune evasion strategy to the virus (Supplementary Fig. S1). Considering the disease severity in S-DEL2 virus-infected farms, the DEL of duad residues in the S1 NTD may be associated with PEDV virulence in neonatal piglets. The S1 NTD is responsible for sialic acid (SA) binding, which facilitates viral entry by recruiting host SA-containing components to invade the thick mucus barrier close to the enterocytes [12]. More interestingly, the pentad \\u0026ldquo;GENQG\\u0026rdquo; at positions 56\\u0026ndash;60 in the S1 NTD was proven to be the major residue for the SA binding activity of the PEDV S protein, and infectious clone-derived mutants with the duad (GE) or pentad (GENQG) DEL displayed the attenuation of SA-dependency and virulence [11]. Because the duad \\u0026ldquo;\\u003csup\\u003e60\\u003c/sup\\u003eGV\\u003csup\\u003e61\\u003c/sup\\u003e\\u0026rdquo;, \\u0026ldquo;\\u003csup\\u003e61\\u003c/sup\\u003eVN\\u003csup\\u003e62\\u003c/sup\\u003e\\u0026rdquo;, or \\u0026ldquo;\\u003csup\\u003e63\\u003c/sup\\u003eST\\u003csup\\u003e64\\u003c/sup\\u003e\\u0026rdquo; residues are located next to or adjacent to the pentad \\u0026ldquo;\\u003csup\\u003e56\\u003c/sup\\u003eGENQG\\u003csup\\u003e60\\u003c/sup\\u003e\\u0026rdquo; in the S1 NTD, it is anticipated that the S-DEL2 variants would weaken the SA-dependent viral entry and diminish the pathogenicity of PEDV. Considering the absence of such field S-DEL2 isolates that can grow in cell culture, the cutting-edge application of reverse genetics (RG) is essential for pursuing loss-of-function research to corroborate the abovementioned aspects. With the availability of the PEDV RG platform in our laboratory [10], our next step is to investigate the precise role of S-DEL2 in the antigenicity, SA dependency, and virulence of PEDV. Nevertheless, our sequence data provide insights into understanding the genetic and pathogenic variation of PEDV field strains. The present study also underscores the need for implementing continuous monitoring and surveillance investigations, followed by swift data sharing and accessibility, to establish PEDV management for preventing future outbreaks/pandemic.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00251145).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edeclarations\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e: The authors declare that they have no conflict of interest.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical approval\\u003c/strong\\u003e: This article does not contain any studies with animals performed by any of the authors.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eJang G, Lee D, Shin S, Lim J, Won H, Eo Y, Kim CH, Lee C (2023) Porcine epidemic diarrhea virus: an update overview of virus epidemiology, vaccines, and control strategies in South Korea. J Vet Sci 24:e58\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchoch CL, Ciufo S, Domrachev M, Hotton CL, Kannan S, Khovanskaya R, Leipe D, Mcveigh R, O'Neill K, Robbertse B, Sharma S, Soussov V, Sullivan JP, Sun L, Turner S, Karsch-Mizrachi I (2020) NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database 2020:baaa062\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLee C (2015) Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virol J 12:193\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLee S, Lee C (2014) Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, 2013. Emerg Infect Dis 20:1223\\u0026ndash;1226\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003ePark J, Lee C (2020) Emergence and evolution of novel G2b-like porcine epidemic diarrhea virus inter-subgroup G1b recombinants. Arch Virol 165:2471\\u0026ndash;2478\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJang G, Park J, Lee C (2021) Successful eradication of porcine epidemic diarrhea in an enzootically infected farm: a two-year follow-up study. Pathogens 10:830\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLee S, Lee DU, Noh YH, Lee SC, Choi HW, Yang HS, Seol JH, Mun SH, Kang WM, Yoo HS, Lee C (2019) Molecular characteristics and pathogenic assessment of porcine epidemic diarrhea virus isolates from the 2018 endemic outbreaks on Jeju Island, South Korea. Transbound Emerg Dis 66:1894\\u0026ndash;1909\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi C, Li W, Lucio de Esesarte E, Guo H, van den Elzen P, Aarts E, van den Born E, Rottier PJM, Bosch BJ (2017) Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies. J Virol 91:e00273\\u0026ndash;e00217\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJang G, Park J, Lee C (2019) Complete genome sequences of novel S-deletion variants of porcine epidemic diarrhea virus identified from a recurrent outbreak on Jeju Island, South Korea. Arch Virol 164:2621\\u0026ndash;2625\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJang G, Lee D, Lee C (2022) Development of a next-generation vaccine platform for porcine epidemic diarrhea virus using a reverse genetics system. Viruses 14:2319\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJang G, Min KC, Lee IH, Won H, Yoon IJ, Kang SC, Lee C (2023) Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets. Vet microbiol 280:109727\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi W, van Kuppeveld FJM, He Q, Rottier PJM, Bosch BJ (2016) Cellular entry of the porcine epidemic diarrhea virus. Virus Res 226:117\\u0026ndash;127\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"archives-of-virology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"arvi\",\"sideBox\":\"Learn more about [Archives of Virology](https://www.springer.com/journal/705)\",\"snPcode\":\"705\",\"submissionUrl\":\"https://submission.nature.com/new-submission/705/3\",\"title\":\"Archives of Virology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4053243/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4053243/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe highly pathogenic-genotype 2b (HP-G2b) porcine epidemic diarrhea virus (PEDV) that caused the 2013–2014 pandemic has evolved in South Korea and has endemically affected the domestic pig industry. This study describes the genotypic traits of novel HP-G2b PEDV strains identified in affected farms experiencing low disease severity with \\u0026lt; 10% neonatal mortality. Nucleotide sequencing revealed common deletion (DEL) patterns of duad residues, termed S-DEL2, at positions 60 and 61, 61 and 62, or 63 and 64 in the N-terminal domain of the spike (S) gene of all isolates. Nevertheless, the S barcode profiles of S-DEL2 variants differed from each other and shared 96.0–99.4% and 98.5–99.6% homology with other South Korean HP-G2b PEDV strains at the S and complete genome levels, respectively. Moreover, genetic and phylogenetic analyses confirmed that the S-DEL2 strains belonged to diverse domestic clades, CK, CK.1, CK.2, or NC. The emergence of novel S-DEL2 strains suggests continuous evolution of PEDV under endemic circumstances, which may result in genetic diversity and distinct clinical presentations. Overall, this study advances our knowledge regarding the genetic and pathogenic heterogeneity of PEDV and emphasizes the significance of active monitoring and surveillance to identify novel variants and further explore their genotypic and phenotypic characteristics.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Genotypic characterization of novel S-DEL variants of porcine epidemic diarrhea virus identified in South Korea\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-04-01 16:21:45\",\"doi\":\"10.21203/rs.3.rs-4053243/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2024-03-30T07:52:42+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-03-27T15:09:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-03-13T04:52:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Archives of Virology\",\"date\":\"2024-03-11T09:50:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"decision\",\"content\":\"Minor Revision\",\"date\":\"2024-03-11T04:32:10+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"archives-of-virology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"arvi\",\"sideBox\":\"Learn more about [Archives of Virology](https://www.springer.com/journal/705)\",\"snPcode\":\"705\",\"submissionUrl\":\"https://submission.nature.com/new-submission/705/3\",\"title\":\"Archives of Virology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"26e41763-1786-4468-b0f4-4e587daabed8\",\"owner\":[],\"postedDate\":\"April 1st, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-05-25T02:55:18+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-04-01 16:21:45\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4053243\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4053243\",\"identity\":\"rs-4053243\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}