Abstract
Background
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a porcine enteric coronavirus that induces watery diarrhea in pigs, causing substantial economic losses in the swine industry. While the molecular and serological epidemiology of SADS-CoV in China has been extensively studied, comprehensive epidemiological studies assessing its prevalence outside China are lacking.
Results
In this study, an indirect enzyme-linked immunosorbent assay based on the SADS-CoV N protein (N-iELISA) was developed to evaluate the seroprevalence of SADS-CoV in Korean pig herds. The optimal conditions for N-iELISA were determined through checkerboard titration of serum samples verified via western blotting. The assay showed sufficient specificity and reproducibility, with a cutoff value of 0.484. A total of 540 field samples collected from pig herds across nine provinces in Korea were subsequently tested using the N-iELISA. The findings revealed an overall seroprevalence of SADS-CoV in Korea of 8.70%.
Conclusions
These results indicate that the N-iELISA is a reliable tool for seroepidemiological studies of SADS-CoV and suggest that the seroprevalence of SADS-CoV in the Korean pig population is relatively low.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12985-025-02834-3.
Keywords: SADS-CoV, Indirect ELISA, Nucleocapsid protein, Seroprevalence, Korea
Background
Swine acute diarrhea syndrome (SADS) is a viral disease affecting pigs caused by swine acute diarrhea syndrome coronavirus (SADS-CoV). It is thought to have originated from bats, specifically the horseshoe bat, which is a known reservoir for various coronaviruses [1–3]. Infected pigs present with severe watery diarrhea, vomiting, rapid dehydration, low appetite, lethargy, and high mortality rates, which can reach 90% in piglets [1]. The primary mode of transmission is the fecal‒oral route, as the virus is shed in high quantities in the feces of infected pigs. SADS can have a substantial impact on the swine industry due to the high mortality rates in piglets, leading to economic losses in regions with significant pork production. Although there is no evidence that SADS-CoV can infect humans, SADS-CoV is considered to have the potential for cross-species transmission because it can infect various cell types, including human primary cells [4, 5]. Experimental studies indicate that the virus can infect other mammals, suggesting a broader host range, but natural infections in animals other than pigs have not been reported [6–8]. SADS-CoV highlights the importance of biosecurity and monitoring in livestock farming, especially considering the potential for new coronaviruses that could impact animal and human health.
The outbreak history of SADS-CoV is relatively recent, with the first known outbreaks occurring in Guangdong Province, southern China, in 2017 [1–3]. From 2017 to 2019, SADS-CoV spread to other provinces in China, such as Fujian, Guangxi, and Henan [9–12]. Despite its potential for widespread damage among swine populations, the virus has thus far remained mostly confined to this region. To date, there have been no confirmed SADS-CoV outbreaks outside China, although concern remains over the potential for spread through the global trade of swine products. Thus, monitoring continues due to the possibility of cross-border spread, as with other swine-related diseases that previously impacted the industry on a global scale.
SADS-CoV is an enveloped positive-sense single-stranded RNA virus that belongs to the genus Alphacoronavirus of the family Coronaviridae. SADS-CoV belongs to the same subgenus as porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) but is genetically distinct, which helps to differentiate it from other coronaviruses affecting pigs [1–3, 13]. The viral RNA is approximately 27.2 kb in length, similar to that of other coronaviruses [14]. The virus has a typical coronavirus structure, with four main structural proteins encoded by the genome: the spike, envelope, membrane (M), and nucleocapsid (N) proteins [13]. Among them, the N protein plays crucial roles in the structure, replication, and immune evasion of the virus [15]. Owing to its high immunogenicity and abundance, the N protein is a major target for diagnostics [16–18].
Given that the symptoms and pathogenesis of SADS-CoV closely resemble those of other porcine coronaviruses, differential diagnosis cannot be made on the basis solely of necropsy findings and must rely on accurate laboratory diagnostics. Currently, several diagnostic methods for SADS-CoV, which fall into two categories, molecular and serological techniques, have been established to detect SADS-CoV infection. Multiplex reverse transcription polymerase chain reaction (RT‒PCR) and multiplex quantitative RT‒PCR (qRT‒PCR) are two commonly used laboratory techniques for diagnosing SADS-CoV and differentiating it from other porcine enteric coronaviruses, such as PEDV, TGEV, and porcine deltacoronavirus (PDCoV) [19–25]. These methods can also distinguish porcine enteric coronaviruses from other circulating porcine viruses. While these approaches are accurate and sensitive, they are also slow, complex, and require specialized equipment and skilled personnel. To simplify and expedite detection, a real-time reverse transcription loop-mediated isothermal amplification test was established [26–28]. A reverse transcription recombinase polymerase amplification fluorescence assay targeting the SADS-CoV M gene was created [29]. Additionally, two sets of primers and a double-quenched probe were designed to target the RNA-dependent RNA polymerase region within the ORF1ab gene for both severe acute diarrhea syndrome coronavirus 2 and SADS-CoV, facilitating the development of a reverse transcription droplet digital PCR assay [30]. The enzyme-linked immunosorbent assay (ELISA) is the most commonly used serological method. ELISA can be employed to detect viruses in clinical samples as well as to identify serum antibodies. An indirect ELISA using the S protein was developed, and its conditions for detecting SADS-CoV antibodies in clinical samples were optimized [31, 32]. Using a monoclonal antibody against SADS-CoV N, Cao et al. developed a blocking and double-antibody sandwich quantitative ELISA to detect SADS-CoV antibodies or viruses [33, 34]. The disease dynamics of SADS-CoV, particularly the humoral immune response, are not yet well understood, but, similar to other coronaviruses, it is thought that IgM initially targets the N protein, followed by IgG targeting both the N and S proteins [35, 36]. These disease dynamics suggest that N protein-specific immune responses are more sensitive for diagnosis, whereas S protein-specific IgG is essential for neutralization and long-term immunity.
Currently, have been no comprehensive investigations into the sero-epidemiology of SADS-CoV in Korea. Considering its geographical location and the introduction of various diseases from China, such as porcine epidemic diarrhea, African swine fever, and severe fever with thrombocytopenia syndrome, it is important to examine the seroprevalence of SADS-CoV in Korea. In this study, a recombinant N protein-based indirect ELISA (N-iELISA) was developed to detect SADS-CoV antibodies. Additionally, the serum prevalence of SADS-CoV in Korea was examined and analyzed using the N-iELISA. These findings provide a potential serological diagnostic tool for SADS-CoV and insights into its seroprevalence in Korea.
Materials and methods
Expression and purification of recombinant SADS-CoV N proteins
The sequence encoding the N protein of the SADS-CoV strain CN/GDWT/2017 (GenBank no. MG557844) was codon-optimized for Escherichia coli (E. coli), synthesized, and cloned into the BamHI-ECoRI restriction sites of the prokaryotic expression vector pET-28a(+) (GenScript) (Fig. 1A, Supplementary Fig. 1). The plasmid pET-28a(+)-SADS-N was transformed into E. coli BL21 cells (Enzynomics, Cat. No. CP111). The transformed E. coli was cultures in Luria Bertani (LB, BioShop, Cat. No. LBL407.500) medium at 37 °C until the optical density unit at 600 nm reached 0.6, and then in LB medium supplemented with 1 mM isopropyl β-D-thiogalactoside (Sigma-Aldrich, Cat. No. I6758) at 15 °C for 16 h. The bacteria were harvested by centrifugation at 4,000 xg for 10 min at 4 °C and lysed in Bugbuster Master Mix (EMD Millipore, Cat. No. 71456-3).
Fig. 1.
Expression and purification of the SADS-CoV N protein. (A) Schematics of the recombinant SADS-CoV N proteins with N-terminal Hig-tag. Intrinsically disordered regions (IDR), the N-terminal domain (NTD) and C-terminal domain (CTD) are shown. (B) SDS‒PAGE analysis of the recombinant SADS-CoV N proteins in total cell lysate, soluble fractions and insoluble fractions. (C) SDS‒PAGE of the purified recombinant SADS-CoV N protein and BSA (as negative control). Western blot analyses of the purified recombinant SADS-CoV N proteins with mouse anti-His antibody. The SADS-CoV N band is indicated by an arrow. The numbers on the left represent the molecular weights of the protein marker (M)
The recombinant N protein was purified by affinity chromatography using ÄKTA (Cytiva) under native conditions. Briefly, the bacteria were harvested by centrifugation at 4,000 xg for 10 min at 4 °C, and resuspended in binding buffer (20 mM Tris, 500 mM NaCl, pH 7.4). After ultrasonication on ice (on 10 s, off 50 s, 50 cycles at 75% amplify), the bacterial lysate was centrifuged at 20,000 xg for 40 min at 4 °C. The supernatant was filtered through 0.45 μm syringe filter and purified through His Trap™ HP (Cytiva, Cat. No. 17524802). The purity was identified by SDS-PAGE, and the immunoreactivity was detected by Western blotting.
Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and Western blotting
Cell lysates and purified proteins were separated by 4–15% gradient SDS-PAGE gels (Bionics, Cat. No. BNROP-0003) and stained with Coomassie Brilliant Blue R250 (Bio-Rad, Cat. No. 1610436). The purified proteins were separated by 12% running gels and separated proteins were then transferred onto polyvinylidene fluoride membranes (Millipore). After being blocked with 5% skim milk (LPS Solution, Cat. No. SKI500) in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at 25 °C, the membranes were incubated overnight at 4 °C with either a mouse monoclonal anti-His antibody (1:1,000, Thermo Fisher Scientific, Cat. No. MA1-21315) or pig plasma (1:100). Following incubation, the membranes were washed three times with TBST for 10 min each and then incubated with either horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, Bioss, Cat. No. BS-0296G-HRP) or anti-swine IgG (1:10,000, Jackson ImmunoResearch, Cat. No. 114-035-003) for 1 h at 25 °C. Protein detection was achieved via the use of an enhanced chemiluminescence reagent (Thermo Scientific, Cat. No. 34580) following the manufacturer’s instructions.
Optimization of the N-iELISA
Ninety-six-well ELISA plates (SPL Life Sciences) were incubated with 100 µl recombinant N protein in ELISA coating buffer (BioLegend, Cat. No. 421701) overnight at 4 °C. The plates were washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and incubated with 400 µl of blocking buffer (5% skim milk in PBST) at 25 °C for 2 h. Next, the plates were incubated with 100 µl of samples diluted with blocking buffer at 25 °C. After three washes with PBST, 100 µl of HRP-conjugated goat anti-swine IgG diluted with blocking buffer was added, and the mixture was incubated at 25 °C. Following another three washes with PBST, 100 µl of tetramethylbenzidine hydrogen peroxidase (TMB) substrate solution (KOMA, Cat. No. K0331082) was added, and the samples were allowed to develop in the dark at 25 °C. The reaction was terminated by the addition of 100 µl of stop solution (KOMA, Cat. No. K0331081). Finally, the optical density at 450 nm (OD450) was immediately measured using a microplate reader (Agilent). All the samples were tested in duplicate and shown as the average.
The optimal conditions for N-iELISA were determined by checkerboard titration. Briefly, gradual dilutions (25, 50, 100 and 200 ng per well) of recombinant N protein was coated on plates. The samples and the HRP-conjugated secondary antibody were serially diluted twofold from 1:50 to 1:400 and from 1:5,000 to 1:100,000, respectively. Blocking buffers with bovine serum albumin (BSA, 1% and 3%, BioShop, Cat. Mo. ALB001.100) and skim milk (1% and 5%) diluted in PBST were tested. Different incubation conditions in blocking buffer were evaluated (1, 2, and 3 h at 25 °C and overnight at 4 °C). The incubation durations with samples (30, 60, 90, and 120 min), the HRP-conjugated secondary antibody (15, 30, 45, and 60 min), and TMB solution (10, 15, 20, and 25 min) were also evaluated. The conditions that resulted in the highest positive/negative sample ratios at OD450 (P/N values) were selected as the optimal reaction conditions.
Cutoff value of the N-iELISA
SADS-CoV negative pig serum and plasma samples (n = 22) were evaluated via N-iELISA to establish the cutoff value. Each sample was tested in duplicate and shown as the average. The cutoff value was determined by calculating the mean OD450 value of SADS-CoV-negative serum samples plus three standard deviations (SDs). Serum samples with OD450 values exceeding this cutoff were classified as SADS-CoV-seropositive.
Repeatability of the N-iELISA
The intra-assay and inter-assay reproducibility of the N-iELISA was determined using SADS-CoV-positive samples (n = 7) and SADS-CoV-negative samples (n = 7). For the intra-assay analysis, each sample was detected in duplicate on the same plate during the same experiment. For the inter-assay assessment, each sample was detected in duplicate on different plates in different experiments. The coefficient of variation (CV) was calculated as the ratio of the SD to the mean OD450 value for each sample.
Cross-reactivity of the N-iELISA with PEDV
To assess cross-reactivity, porcine sera positive for PEDV (n = 9) were analyzed using the N-iELISA. PEDV positive samples were obtained from previous study [37]. Samples with OD450 values exceeding the cutoff were classified as positive.
Evaluation of the clinical performance of the N-iELISA
A total of 540 porcine plasma samples were collected from nine provinces in Korea. These samples were collected at the Animal and Plant Quarantine Agency for disease monitoring in pigs. All animals were healthy and did not show any clinical symptoms such as diarrhea at the time of blood collection. Samples were randomly selected and the number of samples was selected to represent all pigs raised in Korea. Samples with OD450 values exceeding the cutoff were classified as positive.
Statistical analysis
The data were analyzed using Excel and GraphPad Prism 10. All the experiments were independently repeated at least twice to ensure the reproducibility of the data. Data are presented as the mean ± SD. Statistical analysis was performed using the Holm–Sidak multiple Student’s t-test. A P value of < 0.05 was considered statistically significant.
Results
Expression and purification of the recombinant SADS-CoV N protein
A schematic view of the SADS-CoV N with Hig-tag is shown in Fig. 1A. The His-tag was added to confirm the expression and to facilitate affinity purification of the recombinant protein. The synthesized SADS-CoV N gene was expressed in E. coli BL21. The recombinant SADS-CoV N proteins were expressed as a soluble protein with an N-terminal His-tag, yielding a fusion protein of 409 amino acids. SDS-PAGE showed that the His-tagged SADS-CoV N band migrated to the 48 ~ 63 kDa range under reducing conditions (Fig. 1B). The expressed SADS-CoV N proteins were purified using affinity chromatography and analyzed via SDS‒PAGE with Coomassie Brilliant Blue staining and western blotting with an anti-His antibody (Fig. 1C).
Screening SADS-CoV-positive and SARS-CoV-negative samples via Western blotting
SADS-CoV-positive samples were screened via western blotting with recombinant SADS-CoV N proteins using swine plasmas collected in 2023. Seven samples presented a specific band corresponding to the recombinant SADS-CoV N protein (Fig. 2, #1~#7). These samples were used as positive controls when establishing the N-iELISA. Seven negative serum samples collected from sows in 2017 did not recognize the recombinant SADS-CoV N protein [37] (Fig. 2, #8~#14).
Fig. 2.
Validation of swine samples via western blotting. The recombinant SADS-CoV N protein was detected via western blotting using swine plasma (#1~#7) or serum (#8~#14) samples. The SADS-CoV N band is indicated by an arrow. The numbers on the left represent the molecular weights of the protein marker (M)
Establishment of the N-iELISA
The conditions for the N-iELISA were sequentially optimized to maximize the ratio of the SADS-CoV-positive and SADS-CoV-negative sample absorbance values (P/N ratio). First, the antigen concentration was optimized. Sample absorbance showed a dose-dependent positive correlation with antigen concentration ranging from 25 to 200 ng/well, with the highest P/N (P/N ratio = 4.8) observed at 100 ng/well (Fig. 3A). Next, the dilutions of the primary and secondary antibodies and the blocking buffer conditions were evaluated. Among the blocking solutions tested, 5% skim milk provided the best results (Fig. 3B). The optimal dilution for the serum samples (primary antibody) was determined to be 1:200, as it yielded the best P/N value (Fig. 3C). For the secondary antibody, the highest P/N value was achieved at a dilution of 1:100,000 (Fig. 3D). Finally, the reaction times for each step were optimized: blocking (240 min), serum incubation (120 min), secondary antibody incubation (45 min), and TMB substrate reaction (15 min) (Figs. 3E–H).
Fig. 3.
Optimization of the N-iELISA working conditions. Optimization experiments were performed to determine the optimal coating antigen concentration (A), blocking buffer (B), serum sample dilution (C), secondary antibody dilution (D), blocking duration (E), serum incubation duration (F), secondary antibody incubation duration (G), and TMB substrate exposure duration (H). The numbers in the box indicate the positive-to-negative ratio (P/N) values, and the optimal working conditions of N-iELISA are highlighted in bold. All the samples were tested in duplicate and shown as the average. The data are representative of two independent experiments and are presented as the means ± SDs
Cutoff, reproducibility, specificity, and clinical performance of the N-iELISA
To determine the cutoff of the N-iELISA, the sera of twenty-two normal pigs were tested with the developed ELISA (Fig. 4A). The mean OD450 value and SD of these samples were 0.256 and 0.076, respectively. Therefore, the cutoff value for the N-iELISA was determined to be 0.484 (mean ± 3SD). The reproducibility of the N-iELISA was evaluated by determining the intra- and inter-assay CVs. The intra-assay CVs ranged from 0.96 to 7.43% (average = 4.29%), and the inter-assay CVs ranged from 0.41 to 13.63% (average = 4.17%) (Fig. 4B). The variabilities in the average intra- and interassay CVs were less than 10%, suggesting the excellent reproducibility of the N-iELISA. The specificity of N-iELISA was evaluated against sera positive for PEDV because PEDV N showed the greatest amino acid homology with SADS-CoV N. The OD450 values of all tested PEDV-positive sera were lower than the cutoff value (Fig. 4C), indicating that the N-iELISA has no cross-reactivity with PEDV.
Fig. 4.
Determination of the cutoff value, repeatability, and specificity of the N-iELISA. (A) N-iELISA was used to test normal swine serum samples (n = 22). The cutoff value was calculated from the mean OD450 value plus three SDs. (B) SADS-CoV-positive samples (n = 7) and SADS-CoV-negative samples (n = 7) were tested using the N-iELISA, and the CVs were calculated from the OD450 values to determine intra- and inter-assay reproducibility (C) PEDV-positive serum samples were evaluated using the N-iELISA, and the average OD450 value was calculated to determine whether the sample was positive or negative on the basis of the cutoff value. The dashed line indicates the cutoff value. All the samples were tested in duplicate and shown as the average. Results are expressed as the mean at OD450 ± SD values and are representative of at two independent experiments. Statistical significance was assessed using Student’s t-test. ***, P < 0.005
To examine the seroprevalence of SADS-CoV in Korean pig farms, a total of 540 plasma samples from 9 provinces of Korea were collected in 2023 and tested via the N-iELISA. The OD450 values of the clinical samples are shown in Fig. 5; Table 1. The overall seroprevalence of SADS-CoV in Korea was 8.70% (47/540), ranging from 3.33 to 15.00% across the different provinces. Among the 9 provinces, the seroprevalence in Jeonbuk and Jeonnam was the highest at 15.00% (9/60).
Fig. 5.
Evaluation of the clinical performance of the N-iELISA. (A) A total of 540 porcine plasma samples were collected from swine farms across 9 provinces in Korea. The OD450 values were measured using the N-iELISA. All the samples were tested in duplicate and shown as the average. The dashed line indicates the cutoff value. (B) Geographic distribution of SADS-CoV seroprevalences in Korea
Table 1.
Seroprevalence of SADS-CoV in different provinces of Korea
| Province | No. of samples | No. of positive samples | Prevalence (%) |
|---|---|---|---|
| Gyeonggi | 60 | 7 | 11.67 |
| Gangwon | 60 | 1 | 1.67 |
| Chungbuk | 60 | 6 | 10.00 |
| Chungnam | 60 | 2 | 3.33 |
| Kyeongbuk | 60 | 4 | 6.67 |
| Kyeongnam | 60 | 2 | 3.33 |
| Jeonbuk | 60 | 9 | 15.00 |
| Jeonnam | 60 | 9 | 15.00 |
| Jeju | 60 | 7 | 11.67 |
| Total | 540 | 47 | 8.70 |
Discussion
SADS-CoV is a porcine enteric coronavirus that was first reported in 2017 in southern China [1–3]. The clinical symptoms caused by SADS-CoV are undistinguishable from those caused by other porcine enteric coronavirus infections (PEDV, PDCoV, and TGEV) and cannot be differentiated without laboratory-based diagnostic methods [1–3, 31]. Thus far, no significant outbreaks of SADS-CoV have been reported outside China. Serological assays, such as ELISAs, are helpful for monitoring the prevalence of virus infection. Therefore, developing a specific serological diagnostic method is essential. In this study, a N-iELISA was developed and employed to determine the seroprevalence of SADS-CoV in Korean pig farms.
The SADS-CoV N protein is the most conserved protein and plays a pivotal role in packaging viral genomic RNA into long, helical ribonucleoprotein complexes through interactions with the viral genome and M proteins [15]. The N protein is extremely immunogenic and serves as an ideal diagnostic antigen and immunogen because of its low mutation rate and structural stability [16–18]. Consequently, our study selected the N protein as the diagnostic target. The SADS-CoV N protein shares 42.5%, 41.9%, and 16.7% amino acid identity with the N proteins of PEDV, TGEV, and PDCoV, respectively [38], suggesting that there may be cross-reactivity between the N protein of SADS-CoV and these related coronaviruses. Therefore, we experimentally confirmed cross-reactivity with PEDV, which showed the highest similarity to the SADS-CoV N protein (Fig. 3C). Additionally, the N protein was highly conserved, with 93–100% amino acid identity among various SADS-CoV strains. These characteristics demonstrate that the N protein is a highly suitable diagnostic antigen for SADS-CoV via indirect ELISA tests, which is consistent with findings from studies on other coronaviruses [39–43].
There are two common systems for recombinant protein expression: prokaryotic and eukaryotic systems [44]. Prokaryotic expression offers several advantages, including a well-characterized genetic background, the ability to perform high-cell-density fermentation, high production yields, relatively straightforward protein purification processes, and low-cost culture media [45–49]. However, E. coli does not always efficiently express foreign genes, primarily due to differences in codon usage bias between foreign genes and native E. coli genes [50]. Another limitation is that posttranslational modifications in prokaryotic expression systems are incomplete, which may result in lower biological activity of the expressed protein [50]. Unlike highly glycosylated S proteins, N proteins have no N-glycosylation sites; thus, a prokaryotic expression system is suitable for N expression. In addition, to increase expression levels in E. coli, the N sequences were codon optimized.
To the best of our knowledge, this is the first seroepidemiological study on SADS-CoV conducted in Korea. Our results indicate that SADS-CoV antibodies are detected nationwide and the seroprevalence of SADS-CoV in Korea is 8.70%, which is low but significant compared with China. The prevalence of SADS-CoV-positive samples was high in areas adjacent to China and with frequent Chinese travel, such as Gyeonggi, Jeonbuk, Jeonnam, and Jeju. These results imply that SADS-CoV is currently circulating or may have circulated in certain pig herds in Korea. However, direct evidence of SADS-CoV in pig farms could not be established in this study due to the absence of fecal and intestinal sample collection. Furthermore, our prior research, which involved testing fecal samples, also did not detect the presence of SADS-CoV [23]. It is worth noting recent studies from China that report SADS-CoV remains prevalent in Chinese pig herds, particularly in specific provinces [31, 32]. Consequently, more extensive surveillance studies are essential to verify the presence of SADS-CoV and determine its precise prevalence in Korean pig populations.
Typically, a new ELISA diagnostic method must be validated by comparison with an identical or similar commercial ELISA kit or a serum neutralization (SN) test. Although an indirect anti-SADS-CoV IgG ELISA based on the S protein and a blocking ELISA based on the N protein have been developed, no commercial ELISA kit was available for validating the N-iELISA established in this study. Furthermore, since SADS-CoV is not prevalent in Korea, performing an SN test with authentic SADS-CoV samples is difficult. Therefore, the developed ELISA requires further research and comparative studies with other methods to ensure rigorous validation.
In the present study, we used SADS-CoV N proteins associated with His-tag. The His-tag improves protein purity by facilitating efficient purification [51, 52]. In addition, His-tag improve the solubility of recombinant proteins, reducing aggregation and enabling better epitope presentation on ELISA plates [51]. However, if the His-tag is located near or within an immunodominant epitope, it may sterically hinder antibody binding, decreasing sensitivity [53]. And His-tag may sometimes alter protein folding, especially in smaller proteins or peptides, potentially impairing native epitope structure [54]. We added 30 amino acids between the His-tag and the recombinant protein to minimize the effect on the epitope. Future studies are needed to determine whether the presence of the His-tag affects the antigenicity and specificity of the recombinant protein.
Conclusions
In conclusion, an indirect ELISA targeting SADS-CoV N proteins was developed and applied to perform a seroepidemiological survey of SADS-CoV in Korean swine populations. The SADS-CoV N protein, expressed in E. coli, was used as the antigen to detect SADS-CoV without interference from other swine pathogens. The developed ELISA demonstrated high reproducibility and no cross-reactivity. The findings revealed an overall seroprevalence of SADS-CoV in Korea of 8.70%. However, continuous molecular and serological monitoring is still essential due to the geographical proximity of Korea to China, an endemic country of this disease. Additionally, further research on SADS-CoV is crucial, particularly research focusing on the development of antiviral drugs and vaccines to prevent infections in piglets.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We would like to thank Dong-Jun An from the Animal and Plant Quarantine Agency and Hyun-Jin Shin from Chungnam National University for providing the relevant clinical pig samples.
Author contributions
JEP designed experiments and drafted the original manuscript; YJJ and NPL performed the most experiments and data analysis; ESL, MCK, and TKC helped the experiments; JEP supervised the study and provided the resources. All the authors have approved the final manuscript.
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00345532) and by research fund of Chungnam National University.
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
The sample collection for diagnostic and research purposes was carried out by our colleagues from the Animal and Plant Quarantine Agency and Chungnam National University following all relevant national as well as international regulations and according to fundamental ethical principles.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yu-Jeong Jang and Nam Phuong Le are Equally contributed to this article.
References
- 1.Zhou P, Fan H, Lan T, Yang XL, Shi WF, Zhang W, Zhu Y, Zhang YW, Xie QM, Mani S, et al. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of Bat origin. Nature. 2018;556:255–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Gong L, Li J, Zhou Q, Xu Z, Chen L, Zhang Y, Xue C, Wen Z, Cao Y. A new Bat-HKU2-like coronavirus in swine, china, 2017. Emerg Infect Dis. 2017;23:1607–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Pan Y, Tian X, Qin P, Wang B, Zhao P, Yang YL, Wang L, Wang D, Song Y, Zhang X, Huang YW. Discovery of a novel swine enteric alphacoronavirus (SeACoV) in Southern China. Vet Microbiol. 2017;211:15–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Edwards CE, Yount BL, Graham RL, Leist SR, Hou YJ, Dinnon KH 3rd, Sims AC, Swanstrom J, Gully K, Scobey TD, et al. Swine acute diarrhea syndrome coronavirus replication in primary human cells reveals potential susceptibility to infection. Proc Natl Acad Sci U S A. 2020;117:26915–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Yang YL, Qin P, Wang B, Liu Y, Xu GH, Peng L, Zhou J, Zhu SJ, Huang YW. Broad Cross-Species infection of cultured cells by Bat HKU2-Related swine acute diarrhea syndrome coronavirus and identification of its replication in murine dendritic cells in vivo highlight its potential for diverse interspecies transmission. J Virol 2019, 93. [DOI] [PMC free article] [PubMed]
- 6.Chen Y, Jiang RD, Wang Q, Luo Y, Liu MQ, Zhu Y, Liu X, He YT, Zhou P, Yang XL, Shi ZL. Lethal swine acute diarrhea syndrome coronavirus infection in suckling mice. J Virol. 2022;96:e0006522. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Duan Y, Yuan C, Suo X, Li Y, Shi L, Cao L, Kong X, Zhang Y, Zheng H, Wang Q. Bat-Origin swine acute diarrhea syndrome coronavirus is lethal to neonatal mice. J Virol. 2023;97:e0019023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Mei XQ, Qin P, Yang YL, Liao M, Liang QZ, Zhao Z, Shi FS, Wang B, Huang YW. First evidence that an emerging mammalian alphacoronavirus is able to infect an avian species. Transbound Emerg Dis. 2022;69:e2006–19. [DOI] [PubMed] [Google Scholar]
- 9.Zhou L, Li QN, Su JN, Chen GH, Wu ZX, Luo Y, Wu RT, Sun Y, Lan T, Ma JY. The re-emerging of SADS-CoV infection in pig herds in Southern China. Transbound Emerg Dis. 2019;66:2180–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Li K, Li H, Bi Z, Gu J, Gong W, Luo S, Zhang F, Song D, Ye Y, Tang Y. Complete genome sequence of a novel swine acute diarrhea syndrome coronavirus, CH/FJWT/2018, isolated in fujian, china, in 2018. Microbiol Resour Announc 2018, 7. [DOI] [PMC free article] [PubMed]
- 11.Sun Y, Xing J, Xu ZY, Gao H, Xu SJ, Liu J, Zhu DH, Guo YF, Yang BS, Chen XN, et al. Re-emergence of severe acute diarrhea syndrome coronavirus (SADS-CoV) in guangxi, china, 2021. J Infect. 2022;85:e130–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Zhang T, Yao J, Yang Z, Wang J, Yang K, Yao L. Re-emergence of severe acute diarrhea syndrome coronavirus (SADS-CoV) in henan, central china, 2023. Vet Microbiol. 2024;292:110049. [DOI] [PubMed] [Google Scholar]
- 13.Liu C, Huang W, He X, Feng Z, Chen Q. Research advances on swine acute diarrhea syndrome coronavirus. Anim (Basel) 2024, 14. [DOI] [PMC free article] [PubMed]
- 14.Turlewicz-Podbielska H, Pomorska-Mol M. Porcine coronaviruses: overview of the state of the Art. Virol Sin. 2021;36:833–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Jack A, Ferro LS, Trnka MJ, Wehri E, Nadgir A, Nguyenla X, Fox D, Costa K, Stanley S, Schaletzky J, Yildiz A. SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA. PLoS Biol. 2021;19:e3001425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Cong X, Zhang L, Zhu H, Wu M, Zhu Y, Lian Y, Huang B, Gu Y, Cong F. Preparation of a new monoclonal antibody against nucleocapsid protein of swine acute diarrhea syndrome coronavirus and identification of its linear antigenic epitope. Int J Biol Macromol. 2023;239:124241. [DOI] [PubMed] [Google Scholar]
- 17.Han Y, Zhang J, Shi H, Zhou L, Chen J, Zhang X, Liu J, Zhang J, Wang X, Ji Z, et al. Epitope mapping and cellular localization of swine acute diarrhea syndrome coronavirus nucleocapsid protein using a novel monoclonal antibody. Virus Res. 2019;273:197752. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Smits VAJ, Hernandez-Carralero E, Paz-Cabrera MC, Cabrera E, Hernandez-Reyes Y, Hernandez-Fernaud JR, Gillespie DA, Salido E, Hernandez-Porto M, Freire R. The nucleocapsid protein triggers the main humoral immune response in COVID-19 patients. Biochem Biophys Res Commun. 2021;543:45–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Zhu JH, Rawal G, Aljets E, Yim-Im W, Yang YL, Huang YW, Krueger K, Gauger P, Main R, Zhang J. Development and clinical applications of a 5-Plex Real-Time RT-PCR for swine enteric coronaviruses. Viruses 2022, 14. [DOI] [PMC free article] [PubMed]
- 20.Niu JW, Li JH, Guan JL, Deng KH, Wang XW, Li G, Zhou X, Xu MS, Chen RA, Zhai SL, He DS. Development of a multiplex RT-PCR method for the detection of four Porcine enteric coronaviruses. Front Vet Sci. 2022;9:1033864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Ma L, Zeng F, Cong F, Huang B, Huang R, Ma J, Guo P. Development of a SYBR green-based real-time RT-PCR assay for rapid detection of the emerging swine acute diarrhea syndrome coronavirus. J Virol Methods. 2019;265:66–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Pan Z, Lu J, Wang N, He WT, Zhang L, Zhao W, Su S. Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses. Virulence. 2020;11:707–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Baek JH, Lee YM, Vu ND, Kim MH, Zhao J, Le VP, Cho JH, Park JE. A multiplex real-time RT-qPCR assay for simultaneous detection of Porcine epidemic diarrhea virus, Porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus. Arch Virol. 2024;169:82. [DOI] [PubMed] [Google Scholar]
- 24.Zhou H, Shi K, Long F, Zhao K, Feng S, Yin Y, Xiong C, Qu S, Lu W, Li Z. A quadruplex qRT-PCR for differential detection of four Porcine enteric coronaviruses. Vet Sci 2022, 9. [DOI] [PMC free article] [PubMed]
- 25.Si G, Niu J, Zhou X, Xie Y, Chen Z, Li G, Chen R, He D. Use of dual priming oligonucleotide system-based multiplex RT-PCR assay to detect five diarrhea viruses in pig herds in South China. AMB Express. 2021;11:99. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Liu J, Tao D, Chen X, Shen L, Zhu L, Xu B, Liu H, Zhao S, Li X, Liu X et al. Detection of four Porcine enteric coronaviruses using CRISPR-Cas12a combined with multiplex reverse transcriptase Loop-Mediated isothermal amplification assay. Viruses 2022, 14. [DOI] [PMC free article] [PubMed]
- 27.Wang H, Cong F, Zeng F, Lian Y, Liu X, Luo M, Guo P, Ma J. Development of a real time reverse transcription loop-mediated isothermal amplification method (RT-LAMP) for detection of a novel swine acute diarrhea syndrome coronavirus (SADS-CoV). J Virol Methods. 2018;260:45–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Zhou L, Chen Y, Fang X, Liu Y, Du M, Lu X, Li Q, Sun Y, Ma J, Lan T. Microfluidic-RT-LAMP chip for the point-of-care detection of emerging and re-emerging enteric coronaviruses in swine. Anal Chim Acta. 2020;1125:57–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Cong X, Zhu Y, Liu X, Lian Y, Huang B, Luo Y, Gu Y, Wu M, Shi Y. Establishment of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of swine acute diarrhea syndrome coronavirus (SADS-CoV). BMC Vet Res. 2022;18:369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Zhang Z, Wang N, Liu X, Lv J, Jing H, Yuan X, Chen D, Lin X, Wu S. A novel, reverse transcription, droplet digital PCR assay for the combined, sensitive detection of severe acute respiratory syndrome coronavirus 2 with swine acute diarrhea syndrome coronavirus. J AOAC Int. 2022;105:1437–46. [DOI] [PubMed] [Google Scholar]
- 31.Peng P, Gao Y, Zhou Q, Jiang T, Zheng S, Huang M, Xue C, Cao Y, Xu Z. Development of an indirect ELISA for detecting swine acute diarrhoea syndrome coronavirus IgG antibodies based on a Recombinant Spike protein. Transbound Emerg Dis. 2022;69:2065–75. [DOI] [PubMed] [Google Scholar]
- 32.Liu Z, Zhao Y, Yang J, Liu X, Luo Y, Zhu L, Huang K, Sheng F, Du X, Jin M. Seroprevalence of the novel swine acute diarrhea syndrome coronavirus in China assessed by enzyme-linked immunosorbent assay. Front Cell Infect Microbiol. 2024;14:1367975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Cao L, Kong X, Li X, Suo X, Duan Y, Yuan C, Zhang Y, Zheng H, Wang Q. A customized novel blocking ELISA for detection of Bat-Origin swine acute diarrhea syndrome coronavirus infection. Microbiol Spectr. 2023;11:e0393022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Cao L, Kong X, Zhang Y, Suo X, Li X, Duan Y, Yuan C, Zheng H, Wang Q. Development of a novel double-antibody sandwich quantitative ELISA for detecting SADS-CoV infection. Appl Microbiol Biotechnol. 2023;107:2413–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Okba NMA, Muller MA, Li W, Wang C, GeurtsvanKessel CH, Corman VM, Lamers MM, Sikkema RS, de Bruin E, Chandler FD, et al. Severe acute respiratory syndrome coronavirus 2-Specific antibody responses in coronavirus disease patients. Emerg Infect Dis. 2020;26:1478–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Diel DG, Lawson S, Okda F, Singrey A, Clement T, Fernandes MHV, Christopher-Hennings J, Nelson EA. Porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods. Virus Res. 2016;226:60–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Park JE, Shin HJ. Porcine epidemic diarrhea vaccine efficacy evaluation by vaccination timing and frequencies. Vaccine. 2018;36:2760–3. [DOI] [PubMed] [Google Scholar]
- 38.Liu Y, Liang QZ, Lu W, Yang YL, Chen R, Huang YW, Wang B. A comparative analysis of coronavirus nucleocapsid (N) proteins reveals the SADS-CoV N protein antagonizes IFN-beta production by inducing ubiquitination of RIG-I. Front Immunol. 2021;12:688758. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Abdelwahab M, Loa CC, Wu CC, Lin TL. Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to Turkey coronavirus. J Virol Methods. 2015;217:36–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Lugovskaya NN, Scherbakov AV, Yakovleva AS, Tsyvanyuk MA, Mudrak NS, Drygin VV, Borisov AV. Detection of antibodies to avian infectious bronchitis virus by a Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay. J Virol Methods. 2006;135:292–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Saijo M, Ogino T, Taguchi F, Fukushi S, Mizutani T, Notomi T, Kanda H, Minekawa H, Matsuyama S, Long HT, et al. Recombinant nucleocapsid protein-based IgG enzyme-linked immunosorbent assay for the serological diagnosis of SARS. J Virol Methods. 2005;125:181–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Su M, Li C, Guo D, Wei S, Wang X, Geng Y, Yao S, Gao J, Wang E, Zhao X, et al. A Recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against Porcine deltacoronavirus. J Vet Med Sci. 2016;78:601–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Yu F, Le MQ, Inoue S, Thai HT, Hasebe F, Del Carmen Parquet M, Morita K. Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific Recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol. 2005;12:848–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Verma R, Boleti E, George AJ. Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems. J Immunol Methods. 1998;216:165–81. [DOI] [PubMed] [Google Scholar]
- 45.Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol. 2004;22:1399–408. [DOI] [PubMed] [Google Scholar]
- 46.Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev. 1996;60:512–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. 2008;307:249–64. [DOI] [PubMed] [Google Scholar]
- 48.Sorensen HP, Mortensen KK. Advanced genetic strategies for Recombinant protein expression in Escherichia coli. J Biotechnol. 2005;115:113–28. [DOI] [PubMed] [Google Scholar]
- 49.Terpe K. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2006;72:211–22. [DOI] [PubMed] [Google Scholar]
- 50.Fakruddin M, Mohammad Mazumdar R, Bin Mannan KS, Chowdhury A, Hossain MN. Critical factors affecting the success of cloning, expression, and mass production of enzymes by Recombinant E. coli. ISRN Biotechnol. 2013;2013:590587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Waugh DS. Making the most of affinity tags. Trends Biotechnol. 2005;23:316–20. [DOI] [PubMed] [Google Scholar]
- 52.Terpe K. Overview of Tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003;60:523–33. [DOI] [PubMed] [Google Scholar]
- 53.Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci 2013, 73:9 9 1–9 9 23. [DOI] [PMC free article] [PubMed]
- 54.Carson M, Johnson DH, McDonald H, Brouillette C, Delucas LJ. His-tag impact on structure. Acta Crystallogr D Biol Crystallogr. 2007;63:295–301. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
No datasets were generated or analysed during the current study.