Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China

Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China | 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 Short Report Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China Lei Jiang, Lei Wang, Ying Zhao, Jinghui Fan, Yuzhu Zuo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9051150/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract To investigate the current epidemic status of dominant Porcine Epidemic Diarrhea Virus (PEDV) strains and the genetic evolution characteristics of their S proteins in China, as well as to explore strategies for enabling suckling piglets in PED-affected farms to acquire effective maternal antibody protection, this study conducted S gene sequencing on PEDV isolates collected from two epidemic cases, followed by phylogenetic analysis and homology-based three-dimensional modeling. Meanwhile, the titers of IgA and IgG in the PEDV-specific immune response were tracked and analyzed by employing different immunization regimens. The results showed that phylogenetic analysis allowed the identification of distinct variations in the major antigenic epitope CO-26K equivalent (COE) region and the predicted N-glycosylation sites of the S protein. Additionally, the differences in the spatial conformation of the COE region might also account for the inadequate protection of pig herds against G2c strain infection conferred by commercial vaccines that do not contain the G2c strain. Furthermore, feedback immunization combined with inactivated tissue vaccine-based sow booster immunization during gestation could induce high levels of PEDV-specific IgA antibodies, and continuous gestation-stage booster immunization with inactivated tissue vaccines was crucial for maintaining such high antibody titers. Collectively, the findings of this study provide a reference for the establishment of high-level PED maternal antibody protection in pig herds. PEDV Phylogenetic analysis COE IgA IgG N-glycosylation sites Figures Figure 1 Figure 2 Figure 3 Introduction Since 2010, the emergence of new variant strains has led to a nationwide epidemic of PEDV in China. Existing vaccines are basically ineffective against these variant strains, thus dealing a heavy blow to China's swine breeding industry ( Sun D et al. 2016). The spike (S) protein of PEDV binds to host receptors and encompasses immunogenic regions that can elicit neutralizing antibodies (Okda FA et al.2017). The S1 subunit facilitates the virus’s attachment and internalization into host cells, while the S2 subunit includes the fusion peptide (FP), transmembrane domain (TM), and cytoplasmic domain—components that are essential for mediating virus-cell membrane fusion(Wrapp, D et al. 2020; Walls, AC et al. 2020; Du, L et al. 2009). The S1 subunit is made up of the N-terminal domain (NTD) and the CO-26K equivalent (COE) domain (amino acid residues 499–638), which mediates the binding of the virus to host receptors (Chang SH et al. 2002). N-glycans are linked to asparagine residues within specific sequons, namely the consensus sequence Asn-X-Ser/Thr (where X represents any amino acid other than proline)(Chao, Q et al. 2020). Glycans also play a role in regulating viral infectivity, particularly in enhancing the fusion peptide’s capacity to engage with host cells. Viral infection success relies on the conformational transition of the spike protein from its pre-fusion to post-fusion state. The glycan-induced pause creates a critical window for fusion peptides to efficiently engage the host cell membrane-a process that would be markedly inefficient without the presence of glycans. Consequently, the steric characteristics of both the spike protein and glycans may modulate the overall dynamics of host membrane engagement(Pasala C et al. 2024). Since the immune system of newborn piglets is immature and PEDV strains exhibit extremely high variability, sows produce secretory immunoglobulin A (sIgA) through oral immunization with antigens or vaccines, and this antibody is transferred to newborn piglets via milk, thereby protecting the piglets from PEDV infection (Dong SJ et al. 2021; Matías J et al. 2017; Kong F et al. 2023). However, there are currently no research reports on the specific immunization methods to establish high-level maternal antibodies against PEDV. In this study, with the IgA antibody level as the evaluation criterion, different immunization routes and protocols were explored, aiming to provide a reference for the effective prevention and control of PEDV. Materials and methods 1.1 Collection of Clinical Samples Case 1 (HB2024 strain): This farm was an internal self-breeding facility with 6,000 sows, and all weaned piglets were transferred to downstream nursery farms for rearing. No vaccines were administered to the herd prior to the conversion. Case 2 (FJ-NP strain) A large-scale core breeding pig farm in South China had a stock of 4,500 sows. This farm also converted from PEDV-negative to positive. Following the PEDV outbreak, the farm implemented epidemic control measures using feedback feeding combined with tissue-inactivated vaccines. 1.2 PCR and sequencing of collected clinical samples, Phylogenetic analysis and Homology three-dimensional modeling Total RNA was extracted from the diarrheal feces of piglets in Cases 1 and 2 , followed by PCR amplification to obtain the full-length S gene (4161 bp), which was then sent to BGI Genomics for sequencing. The phylogenetic tree was constructed based on 22 sequenced strains(collected in whole China in this study) using Mega. Homology modeling of the S proteins of four strains, namely HB2024, FJ-NP, CV777 and AJ1102, was performed via Swiss-Model with the template 7w6m.1A. The COE region was labeled in Surface mode using PyMOL. DiscoTope-3.0 was utilized to visualize the 3D structures and identify the differences in the 3D conformations of glycosylation sites between HB2024 and AJ1102. 1.3 Tracking and Analysis of PEDV-Specific IgA and IgG Antibodies Four animal experiments were conducted in this study, among which Experiments 1.3.1 to 1.3.3 were carried out on the farm of Case 1 , and Experiment 1.3.4 was performed on the farm of Case 2 . 1.3.1 Comparison of Immune Effects between Feedback Tissue Homogenate and Commercial Vaccine(AJ1102 strain) To investigate the impact of feedback immunization and commercial vaccine immunization on IgA antibody production, 20 sows were randomly selected from the farm at the early stage of the outbreak (prior to July 2022) and divided into a control group and an experimental group, with 10 sows per group. The sows in the experimental group received feedback immunization at 8 weeks before farrowing, and were immunized with the tissue-inactivated vaccine at 3 weeks and 1 week before farrowing. The sows in the control group were immunized with the commercial vaccine at 8 weeks before farrowing, and were also administered the tissue-inactivated vaccine at 3 weeks and 1 week before farrowing. After farrowing, colostrum samples were collected from sows in both groups to determine the IgA antibody levels. 1.3.2 Determination of the Duration of PEDV Mucosal Immunity in Sows To clarify the attenuation period of antibodies induced by the combined immunization strategy of feedback plus tissue-inactivated vaccine, a long-term follow-up monitoring was conducted on the PEDV IgA levels in colostrum of sows across different parities within the immunized herd. Specifically, the colostrum IgA levels of sows in different parities were tested in January 2023, January 2024, and January 2025, respectively. In each year, colostrum samples were collected from 3 sows per parity, with 8 parities included each year, yielding 24 colostrum samples annually. A total of 72 colostrum samples were collected and assayed for IgA antibodies. 1.3.3 Analysis of the Effect of Tissue-Inactivated Vaccine on IgA Levels To verify the necessity of continuous parity-based immunization with tissue-inactivated vaccine in the combined immunization regimen of feedback plus tissue-inactivated vaccine, 20 sows from the farm (which had completed feedback immunization) were selected in July 2022. The colostral antibody levels of these 20 sows in July 2022 were used as the control group. The sows were randomly divided into Test Group 1 and Test Group 2, with 10 sows per group. Sows in Test Group 1 were discontinued from parity-based immunization with tissue-inactivated vaccine, while those in Test Group 2 continued to receive this immunization protocol. After two parities (in May 2023), colostrum samples were collected from the sows in both test groups to determine IgA levels, and comparative analyses were performed on the colostral IgA levels among Test Group 1, Test Group 2 and the control group. 1.3.4 Comparative Tracking Analysis of IgA and IgG Antibodies Ten sows approaching parturition were selected from this herd. Blood samples were collected from the anterior vena cava of the sows before they were moved to the farrowing crates, and the samples were tested for PEDV IgA and IgG antibodies. On the day of farrowing, colostrum samples were collected from these 10 sows. Meanwhile, blood samples were collected from the anterior vena cava of the piglets born to these sows (after the piglets had ingested colostrum). The colostrum samples were tested for IgA antibodies, and the piglet blood samples were tested for IgA and IgG antibodies. 1.3.5 Statistical Analysis All data obtained from antibody detection assays were subjected to statistical analysis and graph plotting using GraphPad Prism 9.0 software. The statistical results were expressed as mean ± standard deviation (SD). *P < 0.05 indicated a significant difference, P < 0.01 indicated an extremely significant difference, and ns indicated no significant difference. 0.8≦|r|≦1: Extremely Strong; 0.5≦|r|<0.8: Strong; 0.3≦|r|<0.5: Moderate; |r|<0.3: no correlation. Results 2.1 Phylogenetic analysis and Homology three-dimensional modeling Phylogenetic tree analysis revealed that both HB2024 (PV389919)and FJ-NP༈PP501550༉, the two strains focused on in this study, were identified as G2c subtype strains. For comparative analysis, the S genes of the PEDV CV777 strain (AF353511) and AJ1102 strain (JX188454) were selected as reference sequences, and their sequences were compared with those of the HB2024 and FJ-NP strains sequenced in this study. In Fig. 1 B, the yellow, blue and green colors represent the trimeric structure of the S protein, while the blue region corresponds to the COE region. Distinct differences can be observed in certain parts of the COE region. The two strains differ in the glycosylation sites within their membrane fusion domains: HB2024 has a β-sheet structure at the relevant region, whereas AJ1102 has a β-turn structure, which lacks one asparagine (Asn) residue (Fig. 2 C). Consequently, AJ1102 has one fewer glycosylation site. 2.2 Results of Tracking and Analysis of PEDV-Specific IgA and IgG Antibodies 2.2.1 Comparison of Immunization Efficacy Between Feedback Tissue Homogenate and Commercial Vaccine As shown in Fig. 3 A, the S/P value of IgA antibodies in the colostrum of piglets in the experimental groups ranged from 8 to 14, while that in the control group ranged from 2 to 4. The S/P value of the experimental groups was extremely significantly higher than that of the control group (P < 0.01). 2.2.2 Effect of Tissue-Inactivated Vaccine on IgA Content Figure 3 B showed that there was no significant difference in the colostral IgA content of Experimental Group 2 after two parities compared with that before two parities (P = 0.075), whereas the colostral IgA content of Experimental Group 1 after two parities was extremely significantly decreased compared with that before two parities (P < 0.01). 2.2.3 Determination of the Duration of Mucosal Immunity in Sows In Fig. 3 C-E, the results showed that the colostral antibody levels of primiparous sows in 2023, sows with 1–4 parities in 2024, and sows with 1–5 parities in 2025 were below or close to the cutoff value of the antibody kit (S/P = 0.5). In contrast, the colostral IgA levels of sows with 2–8 parities in 2023 (Fig. 3 C), sows with 5–8 parities in 2024 (Fig. 3 D), and sows with 6–8 parities in 2025 (Fig. 3 E) were all higher than the cutoff value of the antibody kit (S/P = 0.5). A: Levels of IgA antibody in colostrum of sows treated with different immunizations; B: The Comparison of IgA levels in pigs immunized with and without the inactivated vaccine; C: IgA antibody levels in colostrum of 1–8 parities in Jan 2023; D: IgA antibody levels in colostrum of 1–8 parities in Jan 2024; E: IgA antibody levels in colostrum of 1–8 parities in Jan 2025 2.2.4 Comparative Tracking Analysis results of IgA and IgG Antibodies From Table 1 , there was a very strong correlation between IgG levels in the blood of day-1 piglets and those in the blood of pre-farrowing sows, as well as between IgA levels in the blood of day-1 piglets and colostral IgA levels. Meanwhile, piglets can acquire IgA antibodies against PEDV from the high levels of IgA present in colostrum. Furthermore, there was a moderate correlation between colostral IgA and maternal blood IgG levels, and a strong correlation between maternal blood IgA and IgG levels. Table 1 Variables for comparison(Pearson) of IgG and IgA of FJ-NP strain Items r Correlation Colotrum IgA vs Pre-Farrowing Sows Blood IgA 0.6487 Strong Colotrum IgA vs Pre-Farrowing Sows Blood IgG 0.5260 Moderate Pre-Farrowing Sows Blood IgG vs Pre-Farrowing Sows Blood IgA 0.6188 Strong Pre-Farrowing Sows Blood IgG vs Day 1 Piglets Blood IgG 0.8125 Extremely Strong Day 1 Piglets Blood IgA vs Colotrum IgA 0.8801 Extremely Strong Discussion The results of Case 1 showed that the IgA content in the colostrum of sows immunized with feedback feeding+tissue-inactivated vaccine was extremely significantly higher than that of sows immunized with commercial vaccine + tissue-inactivated vaccine (Fig. 3 A). Feedback feeding enables PEDV to directly infect intestinal epithelial cells, fully activating the mucosal immune system of sows to produce high levels of IgA, thereby providing more effective passive immune protection for piglets. In contrast, although the combination of commercial vaccine and tissue-inactivated vaccine can also induce a certain amount of IgA antibodies, its efficacy is inferior to that of the feedback feeding+tissue-inactivated vaccine regimen. In this experiment, the colostral IgA content of sows with 1–5 parities in 2025 was below the cutoff value of the antibody kit (S/P = 0.5), while that of sows with 6–8 parities was above the cutoff value (S/P = 0.5). This may be attributed to the fact that sows with 1–5 parities in 2025 were only immunized with tissue-inactivated vaccine without feedback feeding acclimation, resulting in IgA content below the cutoff value. The average parity of sows is approximately 2.5 parities per year; thus, sows with 7–8 parities in 2025 were the same group as sows with 5–6 parities in 2024 and 3–4 parities in 2023. These sows (7–8 parities in 2025) could still maintain high IgA levels, indicating that after one effective feedback feeding acclimation and continuous parity-following immunization, the duration of PEDV mucosal immunity in gilts can last up to 5–6 parities. This experiment compared the colostral IgA content of sows receiving continuous parity-following immunization and those discontinuing it. The results showed that after 2 consecutive parities of tissue-inactivated vaccine parity-following immunization, there was no significant difference in sow IgA content compared with that before immunization; however, the IgA content of sows discontinuing parity-following immunization decreased significantly. This indicates that tissue-inactivated vaccine parity-following immunization plays an important role in maintaining high levels of IgA antibodies in sows. Tissue-inactivated vaccines can provide continuous antigen stimulation, activating the secondary immune response mediated by memory B cells, thereby sustaining high-level antibody production. Meanwhile, in Case 2 , there was a moderate correlation between colostral IgA and sow IgG (r = 0.5260), and a strong correlation between sow IgA and IgG (r = 0.6188), which also confirms the positive role of inactivated vaccines in the feedback feeding + inactivated vaccine strategy for PEDV prevention and control. The above experimental results are consistent with the theory proposed by Zhang, E. et al. that after PEDV infection, re-immunization with inactivated vaccine increases the expression of cytokines such as IL-6 and IFN-γ in the intestinal mucosa, further promoting sIgA synthesis and secretion, and proving that re-immunization with inactivated PEDV vaccine can "activate" the intestinal mucosal sIgA response(Zhang E et al. 2020).​ CV777 is a classic vaccine strain of Chinese PEDV, while AJ1102 is a commonly used vaccine strain against variant PEDV(Gao C et al. 2024; Ge FF et al. 2022). Phylogenetic analysis of the PEDV S gene in Cases 1 and 2 showed that both strains belong to the G2c genotype. They differ from AJ1102 (G2b) and CV777 (G1b) in the glycosylation site at position 1196, with an additional glycosylation site (Asn-His-Thr). This may enhance the fusion ability of G2c with host cell membranes, thereby increasing its infectivity. Differences in the spatial conformation of the COE region may also lead to the failure of commercial vaccines (not containing G2c strains) to effectively protect pig herds from G2c strain infection. Currently, no commercial PEDV vaccines containing G2c strains have been approved for marketing in China(Lu X et al. 2025). In summary, this experiment not only sequenced and performed phylogenetic analysis on the PEDV strains outbreak in the two farms, but also identified the differences in the main antigenic epitope COE region and the predicted glycosylation sites of the S protein. In Case 2 , the strong correlation between IgA and IgG in sows further suggests a potential positive role of inactivated vaccines in the feedback feeding + inactivated vaccine strategy adopted for PEDV prevention and control in this case. Meanwhile, by exploring different immunization routes and regimens, this experiment indicated that feedback feeding combined with tissue-inactivated vaccine parity-following immunization is an effective strategy to induce high levels of PEDV IgA antibodies under the experimental conditions, and continuous parity-following immunization with tissue-inactivated vaccine is crucial for maintaining high antibody levels, providing a reference for the effective prevention and control of PED in similar sow farms. With proper gilt acclimation and PEDV inactivated vaccine immunization, sow farms can still maintain excellent production performance. However, when a sow farm needs to supply breeding pigs to other farms, or when market conditions are sluggish, and as an increasing number of sow farms in China are equipped with sub-high efficiency air filters, adopting PED eradication measures is also a favorable choice for sow farms. Declarations Acknowledgments The authors acknowledge PIC(China) customer sow farms for providing the production data and clinical materials in this manuscript. Ethics Statement The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. The research was approved by the ethical review committee at the Hebei Agricultural University (ethical approval number: 2021069). The guidelines of China for the Care and Use of Laboratory Animals were followed. Funding This word was supported by the earmarked fund for Hebei Agriculture Research System (HBCT2024220201, HBCT2024220401). Conflict of Interest Statement The authors declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled ‘Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China’. References Sun D, Wang X, Wei S, et al.(2016) Epidemiology and vaccine of porcine epidemic diarrhea virus in China: a mini-review. J Vet Med Sci 78(3): 355–363. https://doi:10.1292/jvms.15-0446 Okda FA, Lawson S, Singrey A, Nelson J, Hain KS, Joshi LR, et al.(2017) The S2 Glycoprotein Subunit of Porcine Epidemic Diarrhea Virus Contains Immunodominant Neutralizing Epitopes. Virology 509:185–94. https://doi:10.1016/j.virol.2017.06.013. Wrapp, D, Wang, N, Corbett, KS, Goldsmith, JA, Hsieh, CL, Abiona, O (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483), 1260–1263. https://doi:10.1126/science.abb2507 Walls, A.C., Park, Y.J., Tortorici, M.A., Wall, A., McGuire, A.T., and Veesler, D. (2020). Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281–292.e6. https://doi:10.1016/j.cell.2020.02.058 Chang SH, Bae JL, Kang TJ, Kim J, Chung GH, Lim CW, et al.(2002) Identification of the Epitope Region Capable of Inducing Neutralizing Antibodies Against the Porcine Epidemic Diarrhea Virus. Mol Cells 14(2):295–9. https://doi.org/10.1016/S1016-8478(23)15106-5 Chao, Q.; Ding, Y.; Chen, Z.H.; Xiang, M.H.; Wang, N.; Gao, X.D. Recent Progress in Chemo-Enzymatic Methods for the Synthesis of N-Glycans. Front Chem 2020, 8, 513. https://doi.org/10.3389/fchem.2020.00513 Pasala C, Sharma S, Roychowdhury T, Moroni E, Colombo G, Chiosis G.(2024) N-Glycosylation as a Modulator of Protein Conformation and Assembly in Disease. Biomolecules 14(3):282. https://doi:10.3390/biom14030282 Dong SJ, Xie CF, Si FS et al.(2021) Fusheng Si1 Immunization against porcine epidemic diarrhea virus and vaccine development. Chin J Biotechnol 37(8):2603–2613(In Chinese). https://doi:10.13345/j.cjb.200524 Matías J, Berzosa M, Pastor Y, et al.(2017) Maternal vaccination. immunization of sows during pregnancy against ETEC infections. Vaccines 5(4):48. https://doi:10.3390/vaccines5040048 Kong F, Jia H, Xiao Q, et al.(2023) Prevention and Control of Swine Enteric Coronaviruses in China: a review of vaccine development and application. Vaccines 12(1):11. https://doi:10.3390/vaccines12010011 Zhang E, Wang JL, Chen Y et al.(2020) Comparison of oral and nasal immunization with inactivated porcine epidemic diarrhea virus on intestinal immunity in piglets. Exp Ther Med 20: 1596–1606. https://doi:10.3892/etm.2020.8828 Gao C, Chen QY, Hao XX, Wang QS. Porcine Epidemic Diarrhea Virus: Etiology, Epidemiology, Antigenicity, and Control Strategies in China. Animals 2024 14(2):294. https://doi:10.3390/ani14020294. Ge FF, Kang LS, Shen LP(2022) Pathogenicity and Immunogenicity of a Serially Passaged Attenuated Genotype 2c Porcine Epidemic Diarrhea Virus Cultured in Suspended Vero Cells. Front Microbiol. 2022 13:864377. https://doi:10.3389/fmicb.2022.864377 Lu X, Chen C, Wang ZX (2025) Isolation and Characterization of Porcine Epidemic Diarrhea Virus G2c Strains Circulating in China from 2021 to 2024. Vet Sci 12(5):444. https://doi:10.3390/vetsci12050444 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 Mar, 2026 Editor assigned by journal 11 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 06 Mar, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9051150","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":604988563,"identity":"83037b96-83e2-449f-a79b-8caf645c56ab","order_by":0,"name":"Lei Jiang","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Jiang","suffix":""},{"id":604988564,"identity":"c75163a5-f4a9-434b-b79d-d67fcf5cd666","order_by":1,"name":"Lei Wang","email":"","orcid":"","institution":"Binzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wang","suffix":""},{"id":604988565,"identity":"6f005e38-91ac-4e65-aac9-a140922f412b","order_by":2,"name":"Ying Zhao","email":"","orcid":"","institution":"Beijing Ershang Meat Food Group Co.,LTD","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhao","suffix":""},{"id":604988566,"identity":"fe2884b7-5307-453f-b85a-bcd87cce2720","order_by":3,"name":"Jinghui Fan","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jinghui","middleName":"","lastName":"Fan","suffix":""},{"id":604988567,"identity":"b288d86e-6ff4-4c06-8171-05f744a74eb2","order_by":4,"name":"Yuzhu Zuo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDCCAyCi4gCDAZjHRrSWMyRrYWwjRQvfjfRnkj/n3ZEzZz9jwPCh7DAD/+wG/FokbySkSUhue2Zs2ZNjwDjj3GEGiTsH8GsxuJFwTMJw2+HEDQdyDJh52w4zGEgkENKS2CaROOdw/YbzbwyY/xKnJZlN4mDD4QSDG0BbGInRInnmGbNlw7HDhjtnPCs42HMunUfiBgEtfMfTH978UXNY3pw/eeODH2XWcvwzCGhhEEhgkYCxDwAxDwH1QMB/gPkDYVWjYBSMglEwogEAbFZLOBQ+j8AAAAAASUVORK5CYII=","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yuzhu","middleName":"","lastName":"Zuo","suffix":""}],"badges":[],"createdAt":"2026-03-06 13:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9051150/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9051150/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105150294,"identity":"5dd01522-370b-48b0-b04a-9c90e29d0832","added_by":"auto","created_at":"2026-03-22 15:01:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":303026,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree and S protein three-dimensional (3D) homology modeling in Surface mode\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9051150/v1/63a0a48fc9f60fe167c419ee.png"},{"id":105563312,"identity":"7e58b0fa-c683-47c1-b799-e2e3bed809b2","added_by":"auto","created_at":"2026-03-27 12:46:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267027,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Analysis Figure of the N-Glycosylation Site at Position 1196 of the S Protein Between HB2024 and AJ1102 Virus Strains\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9051150/v1/54408a41bb265d7570aae511.png"},{"id":105150293,"identity":"c60d9bbc-657f-4c2d-87ce-08020501f3ed","added_by":"auto","created_at":"2026-03-22 15:01:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":149387,"visible":true,"origin":"","legend":"\u003cp\u003eResults of Tracking and Analysis of PEDV-Specific IgA and IgG Antibodies\u003c/p\u003e\n\u003cp\u003eA: Levels of IgA antibody in colostrum of sows treated with different immunizations; B: The Comparison of IgA levels in pigs immunized with and without the inactivated vaccine; C: IgA antibody levels in colostrum of 1-8 parities in Jan 2023; D: IgA antibody levels in colostrum of 1-8 parities in Jan 2024; E: IgA antibody levels in colostrum of 1-8 parities in Jan 2025\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9051150/v1/8796ead3ab8816a95c2515cc.png"},{"id":105568906,"identity":"61d94ebc-a92c-4f7d-a0a7-759b97d464c7","added_by":"auto","created_at":"2026-03-27 13:10:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1513535,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9051150/v1/1b4e44cf-7c64-41fd-9324-4f19f74f3c2e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince 2010, the emergence of new variant strains has led to a nationwide epidemic of PEDV in China. Existing vaccines are basically ineffective against these variant strains, thus dealing a heavy blow to China\u0026apos;s swine breeding industry ( Sun D et al. 2016).\u003c/p\u003e\n\u003cp\u003eThe spike (S) protein of PEDV binds to host receptors and encompasses immunogenic regions that can elicit neutralizing antibodies (Okda FA et al.2017). The S1 subunit facilitates the virus\u0026rsquo;s attachment and internalization into host cells, while the S2 subunit includes the fusion peptide (FP), transmembrane domain (TM), and cytoplasmic domain\u0026mdash;components that are essential for mediating virus-cell membrane fusion(Wrapp, D et al. 2020; Walls, AC et al. 2020; Du, L et al. 2009). The S1 subunit is made up of the N-terminal domain (NTD) and the CO-26K equivalent (COE) domain (amino acid residues 499\u0026ndash;638), which mediates the binding of the virus to host receptors (Chang SH et al. 2002).\u003c/p\u003e\n\u003cp\u003eN-glycans are linked to asparagine residues within specific sequons, namely the consensus sequence Asn-X-Ser/Thr (where X represents any amino acid other than proline)(Chao, Q et al. 2020). Glycans also play a role in regulating viral infectivity, particularly in enhancing the fusion peptide\u0026rsquo;s capacity to engage with host cells. Viral infection success relies on the conformational transition of the spike protein from its pre-fusion to post-fusion state. The glycan-induced pause creates a critical window for fusion peptides to efficiently engage the host cell membrane-a process that would be markedly inefficient without the presence of glycans. Consequently, the steric characteristics of both the spike protein and glycans may modulate the overall dynamics of host membrane engagement(Pasala C et al. 2024).\u003c/p\u003e\n\u003cp\u003eSince the immune system of newborn piglets is immature and PEDV strains exhibit extremely high variability, sows produce secretory immunoglobulin A (sIgA) through oral immunization with antigens or vaccines, and this antibody is transferred to newborn piglets via milk, thereby protecting the piglets from PEDV infection (Dong SJ et al. 2021; Mat\u0026iacute;as J et al. 2017; Kong F et al. 2023). However, there are currently no research reports on the specific immunization methods to establish high-level maternal antibodies against PEDV. In this study, with the IgA antibody level as the evaluation criterion, different immunization routes and protocols were explored, aiming to provide a reference for the effective prevention and control of PEDV.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Collection of Clinical Samples\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCase 1\u003c/strong\u003e \u003cp\u003e(HB2024 strain): This farm was an internal self-breeding facility with 6,000 sows, and all weaned piglets were transferred to downstream nursery farms for rearing. No vaccines were administered to the herd prior to the conversion.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase 2\u003c/strong\u003e \u003cp\u003e(FJ-NP strain) A large-scale core breeding pig farm in South China had a stock of 4,500 sows. This farm also converted from PEDV-negative to positive. Following the PEDV outbreak, the farm implemented epidemic control measures using feedback feeding combined with tissue-inactivated vaccines.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.2 PCR and sequencing of collected clinical samples, Phylogenetic analysis and Homology three-dimensional modeling\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the diarrheal feces of piglets in Cases \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, followed by PCR amplification to obtain the full-length S gene (4161 bp), which was then sent to BGI Genomics for sequencing. The phylogenetic tree was constructed based on 22 sequenced strains(collected in whole China in this study) using Mega. Homology modeling of the S proteins of four strains, namely HB2024, FJ-NP, CV777 and AJ1102, was performed via Swiss-Model with the template 7w6m.1A. The COE region was labeled in Surface mode using PyMOL. DiscoTope-3.0 was utilized to visualize the 3D structures and identify the differences in the 3D conformations of glycosylation sites between HB2024 and AJ1102.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Tracking and Analysis of PEDV-Specific IgA and IgG Antibodies\u003c/h2\u003e \u003cp\u003eFour animal experiments were conducted in this study, among which Experiments 1.3.1 to 1.3.3 were carried out on the farm of Case \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and Experiment 1.3.4 was performed on the farm of Case \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e1.3.1 Comparison of Immune Effects between Feedback Tissue Homogenate and Commercial Vaccine(AJ1102 strain)\u003c/h2\u003e \u003cp\u003eTo investigate the impact of feedback immunization and commercial vaccine immunization on IgA antibody production, 20 sows were randomly selected from the farm at the early stage of the outbreak (prior to July 2022) and divided into a control group and an experimental group, with 10 sows per group. The sows in the experimental group received feedback immunization at 8 weeks before farrowing, and were immunized with the tissue-inactivated vaccine at 3 weeks and 1 week before farrowing. The sows in the control group were immunized with the commercial vaccine at 8 weeks before farrowing, and were also administered the tissue-inactivated vaccine at 3 weeks and 1 week before farrowing. After farrowing, colostrum samples were collected from sows in both groups to determine the IgA antibody levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e1.3.2 Determination of the Duration of PEDV Mucosal Immunity in Sows\u003c/h2\u003e \u003cp\u003eTo clarify the attenuation period of antibodies induced by the combined immunization strategy of feedback plus tissue-inactivated vaccine, a long-term follow-up monitoring was conducted on the PEDV IgA levels in colostrum of sows across different parities within the immunized herd. Specifically, the colostrum IgA levels of sows in different parities were tested in January 2023, January 2024, and January 2025, respectively. In each year, colostrum samples were collected from 3 sows per parity, with 8 parities included each year, yielding 24 colostrum samples annually. A total of 72 colostrum samples were collected and assayed for IgA antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e1.3.3 Analysis of the Effect of Tissue-Inactivated Vaccine on IgA Levels\u003c/h2\u003e \u003cp\u003eTo verify the necessity of continuous parity-based immunization with tissue-inactivated vaccine in the combined immunization regimen of feedback plus tissue-inactivated vaccine, 20 sows from the farm (which had completed feedback immunization) were selected in July 2022. The colostral antibody levels of these 20 sows in July 2022 were used as the control group. The sows were randomly divided into Test Group 1 and Test Group 2, with 10 sows per group. Sows in Test Group 1 were discontinued from parity-based immunization with tissue-inactivated vaccine, while those in Test Group 2 continued to receive this immunization protocol. After two parities (in May 2023), colostrum samples were collected from the sows in both test groups to determine IgA levels, and comparative analyses were performed on the colostral IgA levels among Test Group 1, Test Group 2 and the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e1.3.4 Comparative Tracking Analysis of IgA and IgG Antibodies\u003c/h2\u003e \u003cp\u003eTen sows approaching parturition were selected from this herd. Blood samples were collected from the anterior vena cava of the sows before they were moved to the farrowing crates, and the samples were tested for PEDV IgA and IgG antibodies. On the day of farrowing, colostrum samples were collected from these 10 sows. Meanwhile, blood samples were collected from the anterior vena cava of the piglets born to these sows (after the piglets had ingested colostrum). The colostrum samples were tested for IgA antibodies, and the piglet blood samples were tested for IgA and IgG antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e1.3.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data obtained from antibody detection assays were subjected to statistical analysis and graph plotting using GraphPad Prism 9.0 software. The statistical results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a significant difference, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 indicated an extremely significant difference, and ns indicated no significant difference. 0.8≦|r|≦1: Extremely Strong; 0.5≦|r|\u0026lt;0.8: Strong; 0.3≦|r|\u0026lt;0.5: Moderate; |r|\u0026lt;0.3: no correlation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Phylogenetic analysis and Homology three-dimensional modeling\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhylogenetic tree analysis revealed that both HB2024 (PV389919)and FJ-NP༈PP501550༉, the two strains focused on in this study, were identified as G2c subtype strains. For comparative analysis, the S genes of the PEDV CV777 strain (AF353511) and AJ1102 strain (JX188454) were selected as reference sequences, and their sequences were compared with those of the HB2024 and FJ-NP strains sequenced in this study. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the yellow, blue and green colors represent the trimeric structure of the S protein, while the blue region corresponds to the COE region. Distinct differences can be observed in certain parts of the COE region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe two strains differ in the glycosylation sites within their membrane fusion domains: HB2024 has a β-sheet structure at the relevant region, whereas AJ1102 has a β-turn structure, which lacks one asparagine (Asn) residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Consequently, AJ1102 has one fewer glycosylation site.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Results of Tracking and Analysis of PEDV-Specific IgA and IgG Antibodies\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Comparison of Immunization Efficacy Between Feedback Tissue Homogenate and Commercial Vaccine\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the S/P value of IgA antibodies in the colostrum of piglets in the experimental groups ranged from 8 to 14, while that in the control group ranged from 2 to 4. The S/P value of the experimental groups was extremely significantly higher than that of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Effect of Tissue-Inactivated Vaccine on IgA Content\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB showed that there was no significant difference in the colostral IgA content of Experimental Group 2 after two parities compared with that before two parities (P\u0026thinsp;=\u0026thinsp;0.075), whereas the colostral IgA content of Experimental Group 1 after two parities was extremely significantly decreased compared with that before two parities (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Determination of the Duration of Mucosal Immunity in Sows\u003c/h2\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E, the results showed that the colostral antibody levels of primiparous sows in 2023, sows with 1\u0026ndash;4 parities in 2024, and sows with 1\u0026ndash;5 parities in 2025 were below or close to the cutoff value of the antibody kit (S/P\u0026thinsp;=\u0026thinsp;0.5). In contrast, the colostral IgA levels of sows with 2\u0026ndash;8 parities in 2023 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), sows with 5\u0026ndash;8 parities in 2024 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), and sows with 6\u0026ndash;8 parities in 2025 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) were all higher than the cutoff value of the antibody kit (S/P\u0026thinsp;=\u0026thinsp;0.5).\u003c/p\u003e \u003cp\u003e A: Levels of IgA antibody in colostrum of sows treated with different immunizations; B: The Comparison of IgA levels in pigs immunized with and without the inactivated vaccine; C: IgA antibody levels in colostrum of 1\u0026ndash;8 parities in Jan 2023; D: IgA antibody levels in colostrum of 1\u0026ndash;8 parities in Jan 2024; E: IgA antibody levels in colostrum of 1\u0026ndash;8 parities in Jan 2025\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Comparative Tracking Analysis results of IgA and IgG Antibodies\u003c/h2\u003e \u003cp\u003eFrom Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, there was a very strong correlation between IgG levels in the blood of day-1 piglets and those in the blood of pre-farrowing sows, as well as between IgA levels in the blood of day-1 piglets and colostral IgA levels. Meanwhile, piglets can acquire IgA antibodies against PEDV from the high levels of IgA present in colostrum. Furthermore, there was a moderate correlation between colostral IgA and maternal blood IgG levels, and a strong correlation between maternal blood IgA and IgG levels.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVariables for comparison(Pearson) of IgG and IgA of FJ-NP strain\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003er\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCorrelation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColotrum IgA vs Pre-Farrowing Sows Blood IgA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColotrum IgA vs Pre-Farrowing Sows Blood IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePre-Farrowing Sows Blood IgG vs Pre-Farrowing Sows Blood IgA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePre-Farrowing Sows Blood IgG vs Day 1 Piglets Blood IgG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExtremely Strong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 1 Piglets Blood IgA vs Colotrum IgA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExtremely Strong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of Case \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed that the IgA content in the colostrum of sows immunized with feedback feeding+tissue-inactivated vaccine was extremely significantly higher than that of sows immunized with commercial vaccine\u0026thinsp;+\u0026thinsp;tissue-inactivated vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Feedback feeding enables PEDV to directly infect intestinal epithelial cells, fully activating the mucosal immune system of sows to produce high levels of IgA, thereby providing more effective passive immune protection for piglets. In contrast, although the combination of commercial vaccine and tissue-inactivated vaccine can also induce a certain amount of IgA antibodies, its efficacy is inferior to that of the feedback feeding+tissue-inactivated vaccine regimen. In this experiment, the colostral IgA content of sows with 1\u0026ndash;5 parities in 2025 was below the cutoff value of the antibody kit (S/P\u0026thinsp;=\u0026thinsp;0.5), while that of sows with 6\u0026ndash;8 parities was above the cutoff value (S/P\u0026thinsp;=\u0026thinsp;0.5). This may be attributed to the fact that sows with 1\u0026ndash;5 parities in 2025 were only immunized with tissue-inactivated vaccine without feedback feeding acclimation, resulting in IgA content below the cutoff value. The average parity of sows is approximately 2.5 parities per year; thus, sows with 7\u0026ndash;8 parities in 2025 were the same group as sows with 5\u0026ndash;6 parities in 2024 and 3\u0026ndash;4 parities in 2023. These sows (7\u0026ndash;8 parities in 2025) could still maintain high IgA levels, indicating that after one effective feedback feeding acclimation and continuous parity-following immunization, the duration of PEDV mucosal immunity in gilts can last up to 5\u0026ndash;6 parities. This experiment compared the colostral IgA content of sows receiving continuous parity-following immunization and those discontinuing it. The results showed that after 2 consecutive parities of tissue-inactivated vaccine parity-following immunization, there was no significant difference in sow IgA content compared with that before immunization; however, the IgA content of sows discontinuing parity-following immunization decreased significantly. This indicates that tissue-inactivated vaccine parity-following immunization plays an important role in maintaining high levels of IgA antibodies in sows. Tissue-inactivated vaccines can provide continuous antigen stimulation, activating the secondary immune response mediated by memory B cells, thereby sustaining high-level antibody production. Meanwhile, in Case \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, there was a moderate correlation between colostral IgA and sow IgG (r\u0026thinsp;=\u0026thinsp;0.5260), and a strong correlation between sow IgA and IgG (r\u0026thinsp;=\u0026thinsp;0.6188), which also confirms the positive role of inactivated vaccines in the feedback feeding\u0026thinsp;+\u0026thinsp;inactivated vaccine strategy for PEDV prevention and control. The above experimental results are consistent with the theory proposed by Zhang, E. et al. that after PEDV infection, re-immunization with inactivated vaccine increases the expression of cytokines such as IL-6 and IFN-γ in the intestinal mucosa, further promoting sIgA synthesis and secretion, and proving that re-immunization with inactivated PEDV vaccine can \"activate\" the intestinal mucosal sIgA response(Zhang E et al. 2020).​\u003c/p\u003e \u003cp\u003eCV777 is a classic vaccine strain of Chinese PEDV, while AJ1102 is a commonly used vaccine strain against variant PEDV(Gao C et al. 2024; Ge FF et al. 2022). Phylogenetic analysis of the PEDV S gene in Cases \u003cspan refid=\"FPar1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e showed that both strains belong to the G2c genotype. They differ from AJ1102 (G2b) and CV777 (G1b) in the glycosylation site at position 1196, with an additional glycosylation site (Asn-His-Thr). This may enhance the fusion ability of G2c with host cell membranes, thereby increasing its infectivity. Differences in the spatial conformation of the COE region may also lead to the failure of commercial vaccines (not containing G2c strains) to effectively protect pig herds from G2c strain infection. Currently, no commercial PEDV vaccines containing G2c strains have been approved for marketing in China(Lu X et al. 2025).\u003c/p\u003e \u003cp\u003eIn summary, this experiment not only sequenced and performed phylogenetic analysis on the PEDV strains outbreak in the two farms, but also identified the differences in the main antigenic epitope COE region and the predicted glycosylation sites of the S protein. In Case \u003cspan refid=\"FPar2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the strong correlation between IgA and IgG in sows further suggests a potential positive role of inactivated vaccines in the feedback feeding\u0026thinsp;+\u0026thinsp;inactivated vaccine strategy adopted for PEDV prevention and control in this case. Meanwhile, by exploring different immunization routes and regimens, this experiment indicated that feedback feeding combined with tissue-inactivated vaccine parity-following immunization is an effective strategy to induce high levels of PEDV IgA antibodies under the experimental conditions, and continuous parity-following immunization with tissue-inactivated vaccine is crucial for maintaining high antibody levels, providing a reference for the effective prevention and control of PED in similar sow farms. With proper gilt acclimation and PEDV inactivated vaccine immunization, sow farms can still maintain excellent production performance. However, when a sow farm needs to supply breeding pigs to other farms, or when market conditions are sluggish, and as an increasing number of sow farms in China are equipped with sub-high efficiency air filters, adopting PED eradication measures is also a favorable choice for sow farms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge PIC(China) customer sow farms for providing the production data and clinical materials in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the ethical policies of the journal, as noted on the journal\u0026rsquo;s author guidelines page, have been adhered to. The research was approved by the ethical review committee at the Hebei Agricultural University (ethical approval number: 2021069). The guidelines of China for the Care and Use of Laboratory Animals were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis word was supported by the earmarked fund for Hebei Agriculture Research System (HBCT2024220201, HBCT2024220401).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled \u0026lsquo;Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China\u0026rsquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun D, Wang X, Wei S, et al.(2016) Epidemiology and vaccine of porcine epidemic diarrhea virus in China: a mini-review. 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Vet Sci 12(5):444. https://doi:10.3390/vetsci12050444\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":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PEDV, Phylogenetic analysis, COE, IgA, IgG, N-glycosylation sites","lastPublishedDoi":"10.21203/rs.3.rs-9051150/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9051150/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e To investigate the current epidemic status of dominant Porcine Epidemic Diarrhea Virus (PEDV) strains and the genetic evolution characteristics of their S proteins in China, as well as to explore strategies for enabling suckling piglets in PED-affected farms to acquire effective maternal antibody protection, this study conducted S gene sequencing on PEDV isolates collected from two epidemic cases, followed by phylogenetic analysis and homology-based three-dimensional modeling. Meanwhile, the titers of IgA and IgG in the PEDV-specific immune response were tracked and analyzed by employing different immunization regimens. The results showed that phylogenetic analysis allowed the identification of distinct variations in the major antigenic epitope CO-26K equivalent (COE) region and the predicted N-glycosylation sites of the S protein. Additionally, the differences in the spatial conformation of the COE region might also account for the inadequate protection of pig herds against G2c strain infection conferred by commercial vaccines that do not contain the G2c strain. Furthermore, feedback immunization combined with inactivated tissue vaccine-based sow booster immunization during gestation could induce high levels of PEDV-specific IgA antibodies, and continuous gestation-stage booster immunization with inactivated tissue vaccines was crucial for maintaining such high antibody titers. Collectively, the findings of this study provide a reference for the establishment of high-level PED maternal antibody protection in pig herds.\u003c/p\u003e","manuscriptTitle":"Genetic Evolution Analysis and Clinical Antibody Tracking Analysis of Two PEDV Strains in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-22 15:01:21","doi":"10.21203/rs.3.rs-9051150/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-12T09:53:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T23:31:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T23:30:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2026-03-06T13:20:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1af90ece-15e8-4a7c-9e81-92454659b0da","owner":[],"postedDate":"March 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T07:38:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-22 15:01:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9051150","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9051150","identity":"rs-9051150","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}