Immunofluorescence assay (IFA)
To evaluate the practicality of the S-iELISA, a total of 111 pig serum samples were evaluate by as above described S-iELISA and IFA, respectively. IFA was conducted as described previously with some modifications (X. Wang et al., 2019; Xu et al., 2019). Briefly, IPI-FX cells (1 × 105) were seeded on 24-well plates and cultured overnight, then infected with SADS-CoV at a multiplicity of infection (MOI) of 0.01. At 24 hours after inoculation, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.2% Triton X-100 for 15 min on the ice. The cells were then blocked with 1% bovine serum albumin (BSA) at 37℃ for 2 h, and incubated with pig sera (1:200; Shenzhen agriculturalproduct Quality and Safety Inspection and Testing Center, China) at 37℃ for 1 h, followed by Cy3-labelled rabbit anti-pig secondary antibody (1:1000; Solarbio, China) at 37℃ for 50 min. The cell nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) [Beyotime Biotechnology (Nanjing, China)] for 5 min at room temperature. After the cells were washed with 1 × PBST, the stained cells were observed with a fluorescence microscope (LEICA DMi8, Germany).
The results of S-iELISA and IFA were compared and the results of IFA were used as the standard for positive and negative to determine the sensitivity and specificity of the S-iELISA as described previously (Lin et al., 2018). Briefly, sensitivity was defined as the ratio of positive tests from the S-iELISA to the positive tests from the reference IFA. Specificity was defined as the ratio of negative tests from the S-iELISA to the negative tests from the reference IFA.
Virus neutralization assays
The neutralizing antibody titers of SADS-CoV in sera were examined according to described previously with some modifications (Li et al., 2015; Wen et al., 2018). Briefly, seven serum samples with different OD450nm values (0.1054, 0.6131, 0.8040, 1.0042, 1.1795, 1.3908, 1.7055) were heated at 56 ℃ for 30 min and were serial 10-fold diluted in serum-free DMEM medium before mixing with an equal volume of 200 TCID50 SADS-CoV strain GDS04 and incubated for 1 h at 37 ℃. A positive control and a negative control were included on each plate. The mixture was added to washed (three times with sterile 1 × PBS) Vero cell monolayers grown in micro-titerplates (Nunc 96-well tissue culture plate, Corning, USA) (100 μL/well) and incubated for 1 h at 37 ℃. Cells were then washed again and incubated in the maintenance medium at 37 ℃ in 5% CO2. After 5 days, CPE was observed using an inverted microscope and the neutralizing concentration was defined as the lowest concentration of antibodies in the serum that prevented the occurrence of cytopathic effect (CPE).
Detection of SADS-CoV, PEDV, TGEV and PDCoV IgG antibodies in field serum samples by ELISA
From July 1 to September 30, 2020, a total of 300 serum samples were collected from commercial growing pigs on different farms with reported diarrhea outbreaks from eleven provinces in the China (Shanxi, Yunnan, Guangdong, Jiangxi, Henan, Hubei, Hebei, Hunan, Qinghai, Anhui, Shanxi), and stored at -20 ℃ until further tested. All pig serum samples were tested for presence of anti-SADS-CoV IgG antibodies using the S-iELISA as above described, and each sample was tested three times. In addition, all pig serum samples also were tested for presence of anti-PEDV (Shenzhen Lvshiyuan Biotechnology Co., Ltd, China), TGEV (Shenzhen Lvshiyuan Biotechnology Co., Ltd, China) and PDCoV (Jianglaibio, China) IgG antibodies using the commercial kits.