The wild-type infectious clone of SADS-CoV was constructed in two steps ( Figure 1A ), with the first step involving the generation of three intermediate constructs, each containing a different region of the genome. For the first intermediate construct, pBeloBAC11-spect-CI-SADS-CoV-WT-F1-F6, six overlapping linear fragments (F1 to F6) mainly comprising ORF1a were generated by RT-PCR, and then using Gibson assembly, the construct was generated with a spectinomycin-selectable gene and the hCMV promoter. The second intermediate construct, pBeloBAC11-spect-SADS-CoV-WT-F7-F10, was assembled from fragments 7 to 10, which contained the entire ORF1b. The third intermediate construct, p15A-amp-SADS-CoV-WT-F11-F14, contained the remainder of the SADS-CoV sequence on plasmid p15A together with an ampicillin resistance gene, and the hepatitis delta virus ribozyme (HDVrz) and simian virus 40 polyadenylation signal (SV40 polyA) sequences. The second step involved releasing the three large fragments from the intermediate constructs by preparative restriction endoenzyme sites. The released fragments were recombined in a chloramphenicol resistance-conferring artificial bacterial chromosome (BAC) vector by Gibson assembly to obtain the infectious clone pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA. The correct construction of the clone was confirmed by restriction enzyme analysis ( Supplementary Figure S1A ).

To generate an infectious deletion mutant for the accessory protein NS3a gene, the PCR-amplified fragment ΔNS3a-amp-ccdB(knockout) was electroporated into Escherichia coli GB08-red-gyrA462 containing pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA to replace the NS3a coding sequence by lambda Red-mediated recombineering. The resulting plasmid, pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA-ΔNS3a-amp-ccdB(knockout), was digested by AscI to remove amp-ccdB and then cyclized by Gibson assembly through the preparative short homologous arms (HA-R). The construct was then transformed into E. coli GB2005 to obtain the infectious clone pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA-ΔNS3a ( Figure 1B , Supplementary Figure S1B ).

To construct the infectious clones for the recombinant SADS-CoV viruses with PEDV antigens, the PCR-amplified fragment ΔNS3a-amp-ccdB(knockin) was electroporated into E. coli GB08-red-gyrA462 containing plasmid pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA. The intermediate construct pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA-ΔNS3a-amp-ccdB(knockin) was then linearized with AscI for incorporation of PEDV(COE+S1D), PEDV(COE+S1D)-Co1, PEDV(COE+S1D)-DCpep, or PEDV(COE+S1D)-Co1-DCpep by Gibson assembly. The constructs were then transformed into E. coli GB2005 to obtain the recombinant vectors pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D), pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep, and pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep ( Figure 1C , Supplementary Figure S1C ).

The coding region of the nucleocapsid (N) gene of SADS-CoV was cloned in the pCI-neo vector as a helper plasmid for viral rescue, and Vero cells were then transfected with the various infectious clones of SADS-CoV accompanied with the helper plasmid pCI-SADS-N; the rescued samples exhibited cytopathic effects in the form of syncytia ( Figure 1D ). The rescued recombinant viruses rSADS-ΔNS3a, rSADS-ΔNS3a-PEDV(COE+S1D), rSADS-ΔNS3a-PEDV(COE+S1D)-Co1, rSADS-ΔNS3a-PEDV(COE+S1D)-DCpep, and rSADS-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep were confirmed by PCR amplification around the NS3a site, with results showing the expected bands of 481bp, 1359 bp, 1401 bp, 1401 bp, and 1452 bp, respectively ( Figure 1E ). Sequencing of the PCR products further validated the correct construction of the rescued recombinant SADS-CoV viruses (rSADS-CoVs).

The pathogenicity of rSADS-ΔNS3a was compared to that of wild-type rSADS-CoV in animal experiments to determine if the NS3a deletion was attenuating ( Figure 2A ). However, neither the NS3a deletion virus nor the wild-type parental virus caused widespread diarrhea in the piglets after infection; only some piglets exhibited mild-to-moderate diarrhea symptoms, which usually resolved quickly. Consequently, the clinical diarrhea scores were generally low across both groups ( Figure 2B ), making it difficult to compare the pathogenicity of the two virus strains based solely on clinical symptoms. Therefore, other parameters, such as weight gain in the piglets and viral shedding, were assessed.

There were no significant differences in weight gain between the rSADS-ΔNS3a group and the rSADS-CoV group, indicating that the NS3a deletion virus and parental virus had similar impacts on piglet growth ( Figure 2C ). Rectal swab samples were collected daily post-inoculation to extract viral RNA, and absolute quantitative fluorescence was used to measure viral nucleic acid copies. Both infection groups exhibited a peak in viral shedding from days 1 to 4 post-inoculation, with no significant differences in the amount or duration of viral shedding between the rSADS-ΔNS3a and rSADS-CoV groups ( Figure 2D ).

On day 4 post-inoculation, three piglets from each group were randomly selected for necropsy. Results showed that the small intestines of piglets in the negative control group had thick walls, while the rSADS-CoV group exhibited significantly thinner and more transparent intestinal walls, with yellow watery stools. In contrast, the rSADS-ΔNS3a group showed milder intestinal lesions, with only partial segments having thinner and more transparent walls. Thus, the intestinal lesions caused by the accessory protein NS3a deletion virus were less severe than those caused by the parental virus. As shown in Figure 2E , no SADS-CoV nucleic acid was detected in the duodenum, jejunum, or ileum of the negative control group. However, in both the rSADS-ΔNS3a and rSADS-CoV groups, virus was detected in the duodenum, jejunum, and ileum, although with no significant differences in overall viral load between the two groups. Histopathological analysis revealed that the two infection groups exhibited varying degrees of small intestinal villus damage compared to the negative control group. The rSADS-CoV group had more severe ileal villus damage, while the rSADS-ΔNS3a group showed only mild villus damage in the small intestine ( Figure 2F ). Therefore, the intestinal tissue damage caused by the NS3a deletion virus was less severe than that caused by the parental virus. The small intestine tissue sections were then subjected to immunohistochemistry (IHC) to detect SADS-CoV N protein. The small intestinal villus epithelial cells of the inoculated piglets were colored to varying degrees by the 3,3’-diaminobenzidine (DAB) chromogen. The rSADS-CoV group showed significant coloration in the jejunum and ileum, while only a small amount of coloration was observed in the duodenum, indicating a higher presence of SADS-CoV antigens in the severely damaged jejunum and ileum. The rSADS-ΔNS3a group showed some coloration in the ileum and jejunum and only a small amount in the duodenum, indicating less presence of SADS-CoV antigens in the jejunum and ileum when compared to the rSADS-CoV group ( Figure 2G ).

The recombinant viruses expressing PEDV antigens were inoculated into Vero cells and continuously passaged to the 20th generation, with stable formation of lesions observed throughout the passaging. Maintenance of the exogenous genes was demonstrated by PCR for generations 2, 5, 8, 11, 14, 17, and 20 ( Figure 3A ). Additionally, sequencing of these PCR products confirmed that the amino acid sequences of the inserted fragments had no mutations, and no compensatory mutations occurred in the SADS-CoV vector sequence even after 20 passages ( Supplementary Figure S2 ). This indicates that the PEDV antigen gene can be stably maintained on the SADS-CoV vector.

The growth trends of the recombinant viruses rSADS-CoV-ΔNS3a-PEDV(COE+S1D), rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep, and rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep, alongside the parental virus rSADS-CoV, were assessed in Vero cells. All four recombinant viruses showed proliferation trends similar to the parental virus, reaching peak titers at 48 hours ( Figure 3B ). In the porcine intestinal epithelial cell line IPI-2I, the three recombinant viruses with targeting peptides showed slightly lower viral copy numbers after 12 hours, indicating slightly lower proliferation levels, when compared to the recombinant virus without targeting peptides and the parental virus ( Figure 3C ). However, the overall growth trends of the four recombinant viruses in IPI-2I cells were fundamentally consistent with that of the parental virus.

The expression of PEDV antigens in the recombinant viruses grown in Vero cells was confirmed through western blot analysis ( Figure 3D ), with results showing target bands of the expected sizes using anti-PEDV spike protein mouse polyclonal antibody, indicating normal expression of the exogenous proteins. Additionally, an immunofluorescence assay (IFA) confirmed the expression of the exogenous proteins in Vero cells inoculated with the recombinant viruses, as evident from the significant fluorescence observed, further validating the normal expression of the inserted protein genes ( Figure 3E ).

The immunogenicity of the recombinant viruses was assessed by inoculating pregnant sows with the viruses ( Figure 4A , Table 1 ). To evaluate the induction of mucosal and cellular immunity by the recombinant viruses in the sows, peripheral blood was collected at parturition to assess the proportions of DC subgroups CD11c and CD80, and T lymphocyte subgroups CD3⁺CD4⁺ and CD3⁺CD8⁺. The percentages of DCs expressing CD80 in the peripheral blood of sows immunized with rSADS-CoV-ΔNS3a-PEDV(COE+S1D), rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep, the inactivated PEDV vaccine, and the negative control group were 5.06 ± 1.08%, 18.32 ± 14.38%, 8.985 ± 4.115%, 8.13 ± 4.37%, 5.215 ± 1.705%, and 4.73 ± 2.47%, respectively ( Figure 4B , Supplementary Figures S3G-L ), and the percentages of DCs expressing CD11c were 22.7 ± 9.4%, 44.95 ± 17.35%, 39 ± 5.1%, 46.95 ± 11.05%, 30.9 ± 12.7%, and 23.4 ± 6.7% respectively ( Figure 4C , Supplementary Figures S3A-F ). The rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep and rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep groups exhibited higher proportions of CD80 and CD11c cells when compared to the inactivated vaccine group and the rSADS-CoV-ΔNS3a-PEDV(COE+S1D) group, with the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1 group showing a significant difference (P ≤ 0.05) in CD11c percentages compared to the negative control group. The percentages of T lymphocyte subgroups CD3⁺CD4⁺ in the immunized and negative control groups were 49.1 ± 5%, 57.15 ± 22.75%, 36.9 ± 9.2%, 49.95 ± 16.45%, 50.05 ± 8.25%, and 29.7 ± 11.2%, respectively ( Figure 4D , Supplementary Figures S3M-R ), and the percentages of CD3⁺CD8⁺ were 17.25 ± 5.25%, 15.08 ± 7.02%, 8.12 ± 2.08%, 16.145 ± 7.655, 14.725 ± 6.975%, and 10.78 ± 5.12%, respectively ( Figure 4E , Supplementary Figures S3M-R ). The rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1 and rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep groups were associated with higher proportions of both CD3⁺CD4⁺ and CD3⁺CD8⁺ cells when compared to the inactivated vaccine group and rSADS-CoV-ΔNS3a-PEDV(COE+S1D) group.

To assess the levels of maternally derived antibodies in piglets from the immunized sows, we utilized ELISAs to measure the levels of PEDV S protein-specific IgG and IgA in the serum of sows in labor and piglets prior to challenge. Compared to the negative control, the serum IgG levels in all experimental groups were elevated, with the recombinant virus-immunized groups exhibiting slightly higher overall IgG levels than the inactivated vaccine group ( Figures 4F, G . Additionally, some piglets in the rSADS-CoV-ΔNS3a-PEDV(COE+S1D) immunized group tested positive for IgA, while IgA levels in other experimental groups did not reach the positive threshold ( Figures 4H, I ). These results indicate that the sows generated antibodies against the PEDV protein and that the antibodies were transferred to the piglets.

To evaluate the clinical protective effects of passive immunization with the recombinant viruses, the piglets were observed daily for clinical symptoms and diarrhea scoring for 8 days post-challenge with PEDV, which was orally administered using 10 mL of 10^6.5 TCID₅₀/mL virus suspension ( Figure 5A ). Results indicated that the negative control group exhibited no illness and accumulated no diarrhea scores, whereas the positive control group displayed severe diarrhea with the highest cumulative diarrhea scores ( Figure 5B ). Diarrhea scores in all recombinant virus-immunized groups were lower than those in the inactivated vaccine group, with the group immunized with the dual-targeting peptide virus rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep showing the lowest cumulative scores, indicating superior clinical protection compared to the other three recombinant viruses ( Figure 5B ).

To validate the results on clinical protection of passively immunized piglets, daily survival rates were recorded post-challenge. The survival rate in the positive control group was 75% post-challenge; however, except for one casualty in the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep-immunized group due to individual piglet weakness, all other immunized groups maintained a 100% survival rate, demonstrating that immunization of pregnant sows with recombinant strains provided substantial immune protection to their nursing piglets ( Figure 5C ).

To assess the impact of PEDV challenge on piglet weights, daily weigh-ins were conducted for 14 days post-challenge. The positive control piglet group exhibited consistent weight loss within 9 days post-challenge, while piglets in all immunized groups showed a slow increase in weight within the first 7 days, with no significant declines ( Supplementary Figure S4A ). Among the recombinant virus-immunized groups, from day 9 post-challenge onwards, the group immunized with the dual-targeting peptide virus rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep experienced the fastest weight gain, indicating better overall condition and suggesting superior clinical protection after immunization with this recombinant virus compared to the other three strains ( Supplementary Figure S4A ).

To evaluate differences in shedding of PEDV among the groups post-challenge, daily rectal swabs were collected for absolute fluorescence quantitative detection of viral RNA (cDNA) copy numbers. Results showed that all groups reached a peak in viral shedding between 3 to 5 days post-challenge, with similar shedding trends post-peak among all immunized groups ( Supplementary Figure S4B ). Compared to the positive control, immunized groups had a shorter duration and lesser amounts of shedding, particularly the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep-immunized group, which had generally lower shedding levels from days 0 to 5 post-challenge, indicating shorter and less impactful effects of PEDV on piglets immunized with this recombinant strain, with quicker recovery observed ( Supplementary Figure S4B ).

On the eighth day following challenge with PEDV, three piglets from each group were randomly selected for necropsy ( Figure 5A ). The piglets in the positive control group exhibited noticeable intestinal wall thinning and transparency, indicating significant pathological changes, whereas no obvious intestinal lesions were observed in piglets from the other groups ( Figure 5D ).

Subsequently, samples of the duodenum, jejunum, and ileum were collected from the necropsied piglets for absolute fluorescence quantitative detection of viral RNA (cDNA) copy numbers for PEDV. The results showed that the PEDV pathogen was present in all sections of the intestine, with the highest viral RNA copy numbers found in the positive control group ( Figure 5E ). Among the immunized groups, the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep group had overall lower viral RNA copy numbers. The copy numbers in the jejunum of piglets immunized with rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1 or rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep were significantly lower (P ≤ 0.05) compared to the positive control group, and notably lower (P ≤ 0.01) in the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep group. Among the four recombinant virus-immunized groups, the three with genomic inserts for targeting peptides showed lower viral RNA copy numbers in the small intestine tissues compared to the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-immunized group, indicating that recombinant viruses with targeting peptides can better protect the small intestine tissues of passively immunized piglets from PEDV infection ( Figure 5E ).

Tissue sections of the duodenum, jejunum, and ileum from the piglets post-viral challenge were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry ( Figure 6 ). The piglets in the positive control group showed significant intestinal tissue damage post-PEDV challenge, with poor integrity of the intestinal villi. In contrast, among the other groups, only the piglets in the rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep-immunized group exhibited severe damage in the jejunum. The intestinal villi of piglets in the other groups were intact, and their intestinal tissues showed no apparent damage ( Figure 6A ). Staining for the PEDV spike protein in the small intestine tissues of the positive control group was pronounced post-PEDV challenge, whereas it was less extensive in the other experimental groups ( Figure 6B ).

This study was carried out in accordance with the recommendations of the National Standards for Laboratory Animals of the People’s Republic of China (GB149258-2010). The protocol was approved by Animal Research Committees of South China Agricultural University. Pigs used for the study were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China.

The SADS-CoV/GDWT/2017 strain (GenBank accession no. MG557844), was isolated in Guangzhou, China, in April 2017. The highly pathogenic GII subtype porcine epidemic diarrhea virus (PEDV) strain PEDV-LN was isolated in Guangzhou, China, in 2018.

African green monkey kidney cells (Vero) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) penicillin-streptomycin. Vero cells were used to rescue SADS-CoV/GDWT/2017-derived recombinants, propagate PEDV, and determine the titer of SADS-CoV/GDWT/2017-derived recombinants.

E. coli GB08-red-gyrA462 was used for deletion of the virulence genes and insertion of genes for antigens. E. coli GB2005 was used for transformation with plasmids constructed by Gibson assembly. pBeloBAC11-spect-ccdB-hyg was used to amplify the linear pBeloBAC11-spect vector for construction of the intermediate plasmids pBeloBAC11-spect-CI-SADS-CoV-WT-F1-F6 and pBeloBAC11-spect-SADS-CoV-WT-F7-F10. p15A-amp-ccdB was used to amplify the linear p15A-amp vector for construction of the intermediate plasmid p15A-amp-SADS-CoV-WT-F11-F14-HDVrz-SV40polyA. pBeloBAC11-cm-ccdB-hyg was used to amplify the linear pBeloBAC11-cm vector for construction of the intermediate plasmid pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA. pCI-amp-ccdB was used as template to amplify the human cytomegalovirus (CMV) immediate-early promoter. R6K-mCMV-GFP-SV40pA-FRT-amp was used as template to amplify SV40polyA. pCI-neo (Promega) was used to amplify the linear pCI-amp vector for construction of the helper plasmid pCI-amp-SADS-CoV-N. p15A-amp-ccdB was used as template to amplify the amp-ccdB resistance gene. The plasmids and bacterial strains used in this study can be freely requested from our laboratory (Shandong University) for academic research.

SADS-CoV genomic RNA was extracted from Vero cells infected with SADS-CoV/GDWT/2017 using an AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Axygen) according to the manufacturer’s instructions. The extracted RNA was then reverse transcribed using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara Bio) according to the manufacturer’s instructions. Fourteen fragments covering the SADS-CoV genome, labeled F1 through F14, were obtained through PCR amplification ( Supplementary Table S1 ). The fragments pBeloBAC11-cm, pBeloBAC11-spect(F1-F6), pBeloBAC11-spect(F7-F10), p15A-amp(F11-F4), CMV, and SV40polyA were obtained through PCR amplification ( Supplementary Table S1 ). After gel electrophoresis, the linear fragments were recovered from the gel and set aside for further use. Additionally, the HDVrz (ribozyme) fragment was synthesized through full gene synthesis and prepared as a water-soluble dry powder for future use.

The pBeloBAC11-spect-CI-SADS-CoV-WT-F1-F6, pBeloBAC11-spect-SADS-CoV-WT-F7-F10, and p15A-amp-SADS-CoV-WT-F11-F14-HDVrz-SV40polyA plasmids were constructed by Gibson assembly using the Gibson Assembly Cloning Kit (NEB). The optimal ratio of the amounts of genomic and vector DNA was determined by titration. After dialyzing, the Gibson-assembled DNA mixture was electroporated into E. coli GB2005. The intermediate plasmids pBeloBAC11-spect-CI-SADS-CoV-WT-F1-F6, pBeloBAC11-spect-SADS-CoV-WT-F7-F10, and p15A-amp-SADS-CoV-WT-F11-F14-HDVrz-SV40polyA were selected on LB plates containing 60 μg/ml spectinomycin or 100 μg/ml ampicillin, and the correct recombinants were identified by PvuII, PstI, or EcoRV restriction enzyme analysis, respectively. The linear fragments CI-F1-F6, F7-F10, and F11-F14-HDVrz-SV40polyA were obtained by removing the vector portion of each intermediate plasmid through digestion at the pre-set NotI+XbaI/EcoRV/EcoRV restriction sites at both ends of the vector. The aforementioned fragments were combined with the PCR-amplified fragment pBeloBAC11-cm using Gibson assembly and transformed into E. coli GB2005 competent cells to obtain the full-length recombinant vector pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA. The recombinants were selected on LB plates containing 15 μg/ml chloramphenicol. The correct recombinants were identified by XmnI restriction enzyme analysis. Sequencing of these plasmids was conducted by Sangon.

The fragments pCI-amp and SADS-CoV-N were obtained through PCR amplification ( Supplementary Table S1 ), and the helper plasmid pCI-amp-SADS-CoV-N was then constructed by Gibson assembly and transformed into E. coli GB2005 competent cells. The recombinants were selected on LB plates containing 100 μg/ml ampicillin, and the correct recombinants were identified by BanI restriction enzyme analysis. Sequencing of the helper plasmid was conducted by Sangon.

The infectious clone and the helper plasmid pCI-amp-SADS-CoV-N were transfected into Vero cells using Lipofectamine 3000 (Thermo Fisher Scientific) to rescue the virus rSADS-CoV.

The fragment ΔNS3a-amp-ccdB(knockout) was obtained through PCR amplification, and the homologous arms with AscI restriction sites were incorporated in the oligonucleotides ( Supplementary Table S1 ). After gel electrophoresis, the linear fragment was recovered from the gel and set aside for further use. Using the infectious clone pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA, the NS3a gene was deleted by Redαβ recombineering in E. coli GB08-red-gyrA462. The recombinants were selected on LB plates containing 100 μg/ml ampicillin and 15 μg/ml chloramphenicol. After confirmation by restriction enzyme analysis, the amp-ccdB segment of the correct clones was removed by AscI digestion. The resulting linear fragment was then combined using Gibson assembly and transformed into E. coli GB2005 to obtain the recombinant plasmid pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA-ΔNS3a. The recombinants were selected on LB plates containing 15 μg/ml chloramphenicol, and the correct recombinants were identified by PstI restriction enzyme analysis and PCR analysis ( Supplementary Table S2 ). Sequencing of this plasmid was conducted by Sangon.

The infectious clone and the helper plasmid pCI-amp-SADS-CoV-N were transfected into Vero cells using Lipofectamine 3000 to rescue the deletion mutant virus rSADS-CoV-ΔNS3a.

PEDV genomic RNA was extracted from Vero cells infected with PEDV using an AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit according to the manufacturer’s instructions. The fragments ΔNS3a-amp-ccdB(knockin) with AscI restriction site, ΔNS3a-PEDV(COE+S1D), ΔNS3a-PEDV(COE+S1D)-Co1, ΔNS3a-PEDV(COE+S1D)-DCpep, and ΔNS3a-PEDV(COE+S1D)-Co1-DCpep were obtained through PCR amplification, and the homologous arms were incorporated in the oligonucleotides ( Supplementary Table S1 ). After gel electrophoresis, the linear fragments were recovered from the gel and set aside for further use. Using the infectious clone pBeloBAC11-cm-CI-SADS-CoV-WT-F1-F14-HDVrz-SV40polyA, the NS3a gene was deleted by Redαβ recombineering in E. coli GB08-red-gyrA462. The recombinants were selected on LB plates containing 100 μg/ml ampicillin and 15 μg/ml chloramphenicol. After confirmation by restriction enzyme analysis, the amp-ccdB segment of the correct clones was removed by AscI digestion. The resulting linear fragment was then combined with linear fragment ΔNS3a-PEDV(COE+S1D), ΔNS3a-PEDV(COE+S1D)-Co1, ΔNS3a-PEDV(COE+S1D)-DCpep, or ΔNS3a-PEDV(COE+S1D)-Co1-DCpep using Gibson assembly and transformed into E. coli GB2005 to obtain the recombinant plasmids pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D), pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep, and pBeloBAC11-SADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep, respectively. The recombinants were selected on LB plates containing 15 μg/ml chloramphenicol, and the correct recombinants were identified by PstI restriction enzyme analysis and PCR analysis ( Supplementary Table S2 ). Sequencing of these plasmids was conducted by Sangon.

The infectious clones and the helper plasmid pCI-amp-SADS-CoV-N were transfected into Vero cells using Lipofectamine 3000 to rescue the viruses rSADS-CoV-ΔNS3a-PEDV(COE+S1D), rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep, and rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep.

The recombinant viruses were inoculated into Vero cells and serially passaged up to the 20th generation, with continuous monitoring of cytopathic effects. RT-PCR was performed on the 2, 5, 8, 11, 14, 17, and 20 passages to confirm the stable presence of the exogenous NS3a gene. A 200 µL sample from the supernatant of each passage of recombinant virus and the positive control rSADS-CoV virus was taken for RNA extraction using a viral DNA/RNA extraction kit (Guangzhou Yueyang Biological Technology Co., Ltd.). The extracted RNA served as a template for amplifying the NS3a gene site using primers SADS-A25-F and SADS-A25-R ( Supplementary Table S2 ) with the HiScript II One Step RT-PCR Kit (Vazyme). The RT-PCR products were analyzed by 1% agarose gel electrophoresis to confirm that the product sizes were correct. Sequencing of these products was conducted by Sangon.

The rSADS-CoV-ΔNS3a-PEDV(COE+S1D) passage 2, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1 passage 2, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep passage 2, and rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep passage 2 viruses, along with the parental virus rSADS-CoV, were inoculated into Vero cells and IPI-2I cells at an MOI of 0.01. Cell supernatants were harvested at 0 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, 30 h, 36 h, 48 h, 60 h, and 72 h, and stored at -80°C. Viral RNA was extracted from a 200 µL sample for each time point using the viral DNA/RNA extraction kit (Guangzhou Yueyang Biological Technology Co., Ltd.). Viral RNA served as a template for quantitative real-time PCR using primers SADS-qF, SADS-qR, and the SADS-probe ( Supplementary Table S3 ) with the HiScript II U+ One Step qRT-PCR Probe Kit to measure viral copy numbers at each time point.

Recombinant viruses were inoculated into Vero cells in six-well plates and incubated at 37°C for 2 h, followed by replacement of the medium with DMEM containing 8 µg/mL trypsin and further incubation at 37°C, 5% CO₂. After 24 h, cells were washed three times with PBS and lysed in 1% protease inhibitor-containing RIPA Lysis Buffer (CWBIO) at 4°C for 30 min. The lysates were collected in 1.5 mL centrifuge tubes, mixed with a quarter volume of 5×SDS loading buffer (CWBIO), and boiled for 10 min to extract total protein. Proteins were separated on a 12.5% PAGE gel using the PAGE Gel Fast Preparation kit (Epizyme) for separation and stacking gels, with electrophoresis at 100 V. Proteins were then transferred to a PVDF membrane using a semi-dry transfer method. The membrane was blocked at room temperature in 5% skim milk in TBST for 2 h, incubated overnight at 4°C with PEDV S mouse pAb diluted 1:1000 in TBST, washed five times with TBST, and then incubated with Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (H+L) (Proteintech, RGAM001), diluted 1:10000 in TBST, at room temperature for 1 h. After washing, the membrane was developed using an Azure 500 imaging system with the Omni-ECL^™ Femto Light Chemiluminescence Kit (Epizyme).

Recombinant viruses were inoculated into Vero cells in 12-well plates and incubated at 37°C for 2 h with DMEM containing 8 µg/mL trypsin. Once cell cytopathy reached 50%, cells were fixed with 4% paraformaldehyde precooled at 4°C for 30 min, and then the cells were permeabilized with 0.1% Triton-X-100 for 15 min at room temperature. After blocking with 5% skim milk in TBST for 1 h, the cells were incubated overnight at 4°C with PEDV S mouse pAb diluted 1:500 in TBST. After washing, cells were incubated with CoraLite488-conjugated Goat Anti-Mouse IgG(H+L) (Proteintech, SA00013-1) diluted 1:500 in TBST for 1 h, washed again, and then stained with 4’,6-diamidino-2-phenylindole (DAPI). Fluorescence microscopy was used to observe and photograph the expression of green and blue fluorescence indicating the presence of exogenous proteins.

Twenty-six 5-day-old piglets, which tested negative for SADS-CoV antibodies, antigens, and other diarrheal pathogen antigens, were selected. Of these, 7 piglets were orally administered 10 mL of 10^-6.8 TCID50/mL rSADS virus suspension, 7 piglets were orally administered 10 mL of 10^-6.8 TCID50/mL rSADS-ΔNS3a virus suspension, and the remaining 5 piglets served as negative controls and were orally administered 10 mL of DMEM. Observations continued for 14 days post-challenge.

Thirty-six pregnant sows, negative for PEDV antibodies and antigens, were selected and divided into six groups of six each. Recombinant viruses rSADS-CoV-ΔNS3a-PEDV(COE+S1D), rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1, rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-DCpep, and rSADS-CoV-ΔNS3a-PEDV(COE+S1D)-Co1-DCpep, along with a commercial inactivated PEDV vaccine and DMEM, were administered via Houhai acupoint injections to the sows 30 days before parturition, with a booster given 9 days before delivery. The negative control group received DMEM. Immunization grouping and doses are detailed in Table 1 . Piglets aged 7 days from groups M1 to M5 and 16 piglets from group M6 (including negative and positive control groups) were orally challenged with PEDV-LN at a dose of 10^7.5 TCID₅₀ per piglet; the negative control group received 10 mL of DMEM per piglet. Observations continued for 14 days post-challenge.

Following the SADS-CoV/PEDV challenge, clinical symptoms and diarrhea scores were observed daily. Clinical symptom scoring criteria for diarrhea were: 0 points for no diarrhea, 1 point for pasty feces, 2 points for loose stool, and 3 points for watery diarrhea/death. Piglets were weighed daily, and cumulative daily weight gain was counted.

At parturition, peripheral blood was collected from the sows, and flow cytometry was used to measure DC subgroups CD11c and CD80, and T lymphocyte subgroups CD3⁺CD4⁺ and CD3⁺CD8⁺. The specific detection steps were as follows: each sample was divided into three groups, with 200 μL of whole blood used for detection in each group. The first group received 10 μL of Mouse Anti-Porcine CD3ϵ-SPRD (PPT3) (SouthernBiotech), 2 μL of Mouse Anti-Porcine CD4-APC (74-12-4) (SouthernBiotech, 4515-11), and 10 μL of Mouse Anti-Porcine CD8α-AF488 (76-2-11) (SouthernBiotech, 4520-30). The second group received 1.5 μL of CD11c/Integrin Alpha X polyclonal antibody (Proteintech, 17342-1-AP), and the third group received 1.5 μL of CD80/B7-1 monoclonal antibody (Proteintech, 66406-1-Ig). The samples were mixed thoroughly and incubated in the dark at room temperature for 20 minutes. Subsequently, 2 mL of 1× hemolysin was added, followed by vortexing and incubation in the dark at room temperature for 20 minutes. The samples were then centrifuged at 1000 r/min for 10 minutes, the supernatant was discarded, and the cells were washed twice with PBS. The cells in the first group were resuspended in 500 μL of PBS for analysis. The second group received 2 μL of FITC-conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (Boster Bio, BA1105), and the third group received 2 μL of Goat Anti-mouse IgG/Alexa Fluor 488 (Bioss, bs-0296G-AF488); samples were then incubated in the dark at 4°C for 30 minutes, followed by two PBS washes, and resuspension in 500 μL of PBS for analysis. The resuspended cells from all three groups were then subjected to flow cytometry analysis using a BD FACSCalibur.

Serum samples were collected from sows in labor and piglets before viral challenge to assess PEDV-specific S protein IgG and IgA levels using the PEDV IgG ELISA kit and PEDV IgA ELISA kit, both obtained from the Guangzhou Yueyang Biological Technology Co., Ltd.

Daily post-challenge, rectal swabs were collected and immersed in centrifuge tubes containing 1 mL of PBS. After vortexing, the swab samples were centrifuged at 10,000 rpm at room temperature for 1 minute. A 200 µL aliquot of the supernatant was used to extract viral RNA using a viral DNA/RNA extraction kit (Guangzhou Yueyang Biological Technology Co., Ltd.). Viral RNA of SADS-CoV/PEDV served as a template for quantitative real-time PCR ( Supplementary Table S3 ), using the protocol for the HiScript II U+ One Step qRT-PCR Probe Kit.

Due to the requirements of tissue sample collection in this experiment, piglets were euthanized using the exsanguination method following anesthesia to minimize the effect of blood on the tissue samples. Zoletil^®50 (Virbac) was diluted according to the manufacturer’s instructions and administered intramuscularly at a dose of 20 mg/kg body weight. After the piglets became unconscious, with relaxed muscles, stable breathing, and the absence of corneal reflex, the carotid artery was severed using a scalpel to induce exsanguination and euthanasia. Four days after SADS-CoV challenge and eight days after PEDV challenge, three piglets from each group were randomly selected for necropsy after euthanasia by the method described above to document and photograph intestinal lesions. Samples from the duodenum, jejunum, and ileum were collected. Each tissue sample, weighing 0.5 g, was placed in a grinding tube with 1 mL PBS and grinding beads, and homogenized. After centrifugation at 10,000 rpm for 2 minutes, 200 µL of the supernatant was extracted using the viral DNA/RNA extraction kit. Viral RNA of SADS-CoV and PEDV served as a template for quantitative real-time PCR ( Supplementary Table S3 ), following the protocol for the HiScript II U+ One Step qRT-PCR Probe Kit to determine viral copy numbers.

Samples of 1-2 cm in size from the duodenum, jejunum, and ileum of each group were fixed in 4% formaldehyde solution for 48 hours, and then embedded in paraffin, sectioned, and subjected to H&E staining. Dehydration was performed stepwise using low to high concentrations of alcohol, followed by embedding in paraffin and further processing and then preparation of tissue sections using standard procedures. The sections were stained with Harris hematoxylin for 3-5 minutes, rinsed in tap water for 1-2 minutes, differentiated in 0.8%-1% hydrochloric acid alcohol, and rinsed again in tap water. Bluing was performed in a diluted lithium carbonate solution, followed by rinsing for 1-2 minutes, and then staining in eosin (alcohol-soluble) for 1-2 seconds. Dehydration was performed by immersing the sections in 95% ethanol and absolute ethanol for 1-2 minutes each. The sections were then cleared in xylene, mounted, and scanned with a brightfield scanner.

Separate sections of the above paraffin-embedded samples were processed in synchrony for IHC. Paraffin-embedded sections were deparaffinized three times with an eco-friendly deparaffinizing agent for 10 minutes each and then rehydrated through a graded series of ethanol: absolute, 95%, and 75% for 5 minutes each. Samples were washed three times with Tris-buffered saline (TBS), 3 minutes per wash, and then citrate buffer (pH 6.0) was added prior to further processing by boiling, cooling, and then washing three times with TBS, 5 minutes per wash, before incubation in 3% hydrogen peroxide at room temperature for 30 minutes to block endogenous peroxidase activity. Samples were then rinsed three times with TBS, 5 minutes per wash, placed in TBST, and then blocked with 10% goat serum at room temperature for 30 minutes. After discarding the serum, approximately 50 µl per section (dependent on tissue size) of primary antibody (anti-PEDV S protein mouse polyclonal antibody), which was diluted 1:1000 in 10% goat serum, was added followed by incubation overnight at 4°C. After washing three times with TBST, goat anti-mouse secondary antibody was added followed by incubation at 37°C for 45 minutes, and washing again three times with TBST. After discarding the TBST, 50 µl of freshly prepared DAB was added per section (dependent on tissue size), and the reaction was terminated with tap water after an appropriate period of time. Samples were then processed by staining with hematoxylin and bluing solution to colour the nuclei and then mounting using standard protocols prior to scanning with a brightfield scanner.

Differences between groups were analyzed using GraphPad Prism 8.0 software. Experimental data were presented as mean ± standard error of the mean (SEM). A p-value > 0.05 was considered “not significant”, p ≤ 0.05 was considered “significant” (denoted by *), and p ≤ 0.01 was considered “highly significant” (denoted by **).