A total of 12,978 clinical porcine serum samples were collected between 2022 and 2023 across 29 provinces of China by the testing center of Wuhan Keqian Biological Co. Ltd. and stored for future use ( Table 1 ). The SADS-CoV-positive serum was provided by the Wuhan Institute of Virology, Chinese Academy of Sciences. Furthermore, our laboratory stored 81 Specific Pathogen Free (SPF) swine sera. Additionally, our laboratory also preserved other porcine pathogen-positive serum samples including 4 PEDV, 5 TGEV, 5 African swine fever virus (ASFV), 5 porcine circovirus-2 (PCV-2), 6 porcine pseudorabies virus (PRV), 8 porcine reproductive and respiratory syndrome virus (PRRSV), 4 foot-and-mouth disease virus (FMDV), 3 porcine polioviruses (PPV), 3 Japanese encephalitis virus (JEV), and 6 classical swine fever viruses (CSFV).

Huh-7 cells were cultured in Dulbecco’s Minimal Essential Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS,LifeTechnologies) and 1% (w/v) antibiotics (penicillin,streptomycin;Invitrogen); Chinese hamster ovary (CHO) suspension cells were cultured using serum-free medium (Eminence). The SADS-CoV virus strain was isolated from piglet samples from the 2017 outbreak of piglet diarrhea in Guangdong, China, and preserved in the Microbial Virus Collection Center of Wuhan Institute of Virus Research, Chinese Academy of Sciences.

The SADS-CoV N gene was synthesized (Tsingke Biotechnology Co., Ltd.) and inserted into the PET-28a-His vector for prokaryotic expression. The modified vector was transformed into E. coli BL21 (DE3) cells, which were cultured overnight at 16°C in medium containing 1 mM IPTG. Bacteria were collected by centrifugation, resuspended in PBS, and fragmented by sonication. The N-terminal region, comprising 1–543 amino acids (SADS-CoV S1) of the SADS-CoV S protein containing the signal peptide, was synthesized and cloned into the pCMV-His expression vector to construct the recombinant plasmid pCMV-S1-His. The recombinant plasmid pCMV-S1-His was transfected into serum-free CHO cells using the polyetherimide (PEI) transfection reagent and cultured for 1 week. The supernatant was filtered through a 0.45 µm filter, collected, and purified with AKTA Pure 25 using Ni resin at 4°C. Proteins were subjected to gel filtration chromatography using a Superdex 200 Increase 10/300 GL column, and the protein concentration was determined using a G250 staining solution (Beyotime, Shanghai, China), following the manufacturer’s protocol.

The purified recombinant SADS-CoV N and S1 proteins was analyzed via 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane, which was blocked with 1% BSA in TBST for 1 h at 37°C. The nitrocellulose membranes were then incubated overnight with SADS-CoV-positive or negative serum as the primary antibody at 4°C. After incubation, the nitrocellulose membrane was washed thrice with TBST buffer for 10 min each and incubated with goat anti-swine enzyme-labeled secondary antibody (SouthernBiotech, USA) for 1 h at 37°C. Finally, the protein bands were detected using the ECL system (Advansta, San Jose, CA, USA).

Specifically, 96-well ELISA plates were coated with 100 μL purified recombinant S1 protein in bicarbonate buffer (pH = 9.6) overnight at 4°C. The plates were washed thrice with PBST (PBS containing 0.05% Tween-20) and blocked in PBST containing 2% BSA for 2 h at 37°C. After washing the plates thrice, 100μL pig serum sample diluted with sample diluent (PBST containing 2% BSA) was added and incubated for 1 h at 37°C. After washing with PBST five times, 100 μL horseradish peroxidase (HRP)-conjugated goat anti-swine IgG (SouthernBiotech, USA) diluted with secondary antibody protectant was added to the plates and incubated at 37°C for 30 min. After washing with PBST five times, 100 µL tetramethylbenzidine hydrogen peroxide (TMB) substrate solution was added to each well, and the color was developed in the dark at 37°C for 10 min, followed by the addition of 50 µL of stop solution. Finally, the reading was immediately recorded at 630 nm optical density using a microplate reader (TECAN, Switzerland).

The optimal dilution of recombinant protein S1 with enzyme-labeled secondary antibody was determined using the checkerboard method, in which the concentration of S1 protein was gradually diluted in the following sequence: 4, 2, 1, 0.5, and 0.25 µg/mL. The enzyme-labeled secondary antibody was serially diluted two-fold from 1:5000 to 1:40,000. The ratio of positive serum OD₆₃₀ value to negative serum OD₆₃₀ value (P/N value) was maximized as the best reaction condition. Simultaneously, the conditions of ELISA for the detection of porcine serum antibodies, such as serum sample dilution and incubation time, were optimized.

Huh-7 cells (2.5×10⁵) were inoculated in 12-well plates, cultured overnight, and then infected with SADS-CoV at an MOI of 0.1. After incubation at 37°C for 24 h, cells infected with SADS-CoV were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.01% Triton X-100 on ice for 15 min. Then 1% BSA was added to the cells such that they were submerged and incubated for 30 min, followed by incubation with ELISA-positive serum or ELISA-negative serum (1:50) 2 h at 37°C, which was followed by incubation with FITC-conjugated goat anti-porcine secondary antibody (1:1000) (SouthernBiotech, USA) for 1 h at 37°C. Cells were incubated with 4,6-diamidino-2-phenylindole (DAPI;1:1000) for 10 min, and the stained nuclei were visualized under a fluorescence microscope (Leica, German).

Data were analyzed using GraphPad Prism 8 and Excel. All experiments were repeated at least thrice.

SADS-CoV mediates receptor binding of the virus to host cells through the RBD of the S1 subunit of the S protein, and studies on the receptor-binding region of SADS-CoV are lacking. Therefore, we selected the S1 subunit of the SADS-CoV S protein as the target for diagnostic test development. A schematic view of the SADS-CoV S1 protein is shown in Figure 1A . The optimized signal peptide sequence was constructed at the N-terminus to ensure high secretion efficiency of the protein, and a His-tag was attached at the C-terminus to facilitate affinity purification of the protein. Recombinant SADS-CoV S1 was successfully expressed in CHO cell culture supernatants, and SDS-PAGE showed that the S1-His band migrated to the 100–130 kDa range under reducing conditions ( Figure 1B ). Western blotting results showed that the recombinant S1-His protein expressed via CHO cells could react with SADS-CoV-positive serum but not with SADS-CoV-negative serum ( Figures 1C, D ), indicating that the recombinant S1-His protein has excellent immunogenicity.

The assay conditions of the S1-iELISA were determined by checkerboard titration to maximize the SADS-CoV-positive to SADAS-CoV-negative serum ratio (P/N). The results showed that the dilution of the coating antigen S1 was 1 µg/mL, and the dilution of the enzyme-labeled secondary antibody was 1:20,000 when the P/N ratio was at the maximum ( Figures 2A, B ). Subsequently, we optimized the dilution of the serum samples and showed that the best discrimination between positive and negative serum samples was achieved when they were diluted 1:100 ( Figure 2C ). Finally, we optimized the reaction times of serum, enzyme-labeled secondary antibody, and tetramethylbenzidine hydrogen peroxide (TMB) substrate solution to achieve the best assay conditions. As shown in Figures 2D–F , the optimal reaction times for serum, enzyme-labeled secondary antibody, and TMB substrate solutions were 60 min, 30 min, and 10 min, respectively, when the ELISA conditions were optimized.

In total, 81 porcine-negative serum samples were used to determine the cut-off value for the S1-iELISA. As shown in Figure 3A , the mean OD₆₃₀ value of these negative samples was 0.2057, and the standard deviation (SD) was 0.0742. Therefore, the cut-off value for the S1-iELISA was 0.4282 (Mean+3SD), which indicates that SADS-CoV-positive sera were well distinguished from SADS-CoV-negative sera when the cut-off value was 0.4282.

The stability of S1-iELISA was assessed by determining intra- and inter-assay coefficient of variation (CV). The intra-assay CV of seven positive samples and one negative sample ranged from 0.21% to 7.12%; the inter-assay CV ranged from 0.35% to 7.9% ( Figure 3B ). Both intra-assay and inter-assay variabilities in the experiments were less than 10%, indicating the favorable reproducibility of S1-iELISA.

To identify the specificity of the S1-iELISA, positive sera for common porcine pathogens were assayed, and the mean OD₆₃₀ values of PEDV-, TGEV-, ASFV-, PCV-2-, PRV-, PRRSV-, FMDV-, PPV-, JEV-, and CSFV-positive samples were 0.3065, 0.3085, 0.1925, 0.1753, 0.1874, 0.2323, 0.1272, 0.2989, 0.3113, and 0.1539, respectively ( Figure 3C ). The OD₆₃₀ values of these positive pathogen sera were all below 0.4282, indicating that these serum samples were SADS-CoV-seronegative and non-cross-reactive with this S1-iELISA. Therefore, the SADS-CoV S1-iELISA had excellent specificity.

To further validate the specificity of the S1-iELISA, we randomly selected five ELISA-positive sera samples and one ELISA-negative serum sample that had been tested by the S1-iELISA as primary antibodies for indirect immunofluorescence assay and western blotting. The IFA results showed that cells infected with SADS-CoV did not fluorescence when incubated with ELISA-negative serum, and cells infected with SADS-CoV showed green fluorescence when incubated with ELISA-positive serum ( Figure 4A ). In addition, western blotting results showed that the recombinant SADS-N protein displayed bands when incubated with ELISA-positive sera but not when incubated with ELISA-negative sera ( Figure 4B ). The above results indicate that S1-iELISA has excellent specificity.

In this study, 12,978 swine clinical serum samples were collected from 29 provinces/autonomous regions/municipalities in China between 2022 and 2023, and the general seroprevalence of SADS-CoV in China was 59.97% as determined using S1-iELISA ( Table 1 ; Figure 5A ). Among the 29 provinces/autonomous regions/municipalities where samples were collected, the seroprevalence ranged from 16.7% to 77.12% in different provinces ( Table 1 ) and from 42.61% to 68.45% in different months ( Figure 5A ). The seroprevalence in Northeast China, North China, Northwest China, Central China, East China, Southwest China, and South China was 66.7% (587/880), 63.67% (1360/2136), 57.3% (621/1083), 62.02% (1556/2509), 56.42% (1626/2882), 58.39% (1239/2122), and 58.13% (794/1366), respectively ( Figure 5B ). The above results indicate that SADS-CoV is widely prevalent in Chinese swine herds, and that the seroprevalence of SADS-CoV is higher in Northeast, North, and Central China than in other regions. The seroprevalence rates of SADS-CoV in spring, summer, autumn, and winter were 62.19% (2158/3470), 61.24% (2313/3777), 56.64% (1552/2740), and 58.84% (1760/2991), respectively ( Figure 5C ). Compared to that in other seasons, SADS-CoV seroprevalence was the highest in spring, with a prevalence of 62.19%, and the lowest in fall, with a prevalence of 56.64%. Therefore, season may be a key factor influencing the prevalence and outbreak of SADS-CoV in Chinese swine herds.