Cloning, expression and purification of SVA VP1, VP2, and VP3 protein
The full length gene sequences for SVA VP1, VP2, and VP3 from strain 11-55910-3 (Genbank ID AGM16001) was optimized for expression in E. coli, synthesized (Integrated DNA Technologies, Inc., Coralville, IA), and cloned into a modified pET24b vector (Novagen, Madison, WI) [12] using the In-Fusion cloning kit (Clontech, Mountain View, CA) following the manufacturer’s directions. Gene sequences for VP1, VP2, and VP3 antigens were confirmed by sequencing. Protein expression and purification was performed as previously described [13]. Purity was analyzed by SDS-PAGE, stained using Imperial™ protein stain (ThermoFisher Scientific, Waltham, MA). Protein concentrations used to establish plate coating conditions were determined using the Quick-start Bradford protein assay following manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA) using a Bio-tek Epoch plate reader (BioTek Instruments, Inc., Winooski, VT).
Serum samples
Recent and archived porcine serum samples that had been previously submitted to the University of Minnesota Veterinary Diagnostic Laboratory (UMN-VDL) for routine diagnostics or specific viral pathogen evaluation were obtained. The samples were provided for diagnostic purposes, not specifically for use in this study. In addition, positive serum samples were obtained from 34 sows clinically diagnosed with vesicular lesions and bled periodically over a 60-day period starting at the first observation of clinical signs (sampling at day 0, 4, 11,18, 25, 39, and 60) (n = 205). Serum samples that tested negative for SVA by PCR (n = 116) were obtained from sows and finishing pigs from various farms with no prior evidence of vesicular disease. These animals were assumed to be SVA antibody-negative and were treated as such in this study. Porcine epidemic diarrhea virus (PEDV) seropositive samples (n = 40) were archived samples from our laboratory and encephalomyocarditis virus (EMCV) seropositive samples were obtained from the UMN-VDL (n = 8). FMD seropositive samples (n = 21) were archived samples from Plum Island Animal Disease Center representing 8 different serotypes, 2 field samples, and ranged from 0 to 36 days post-infection/post-challenge. The FMDV samples were examined for cross-reactivity against the SVA VP1, VP2, and VP3 ELISA at Plum Island Animal Disease Center (PIADC) facilities following the ELISA protocol described here.
Antibody detection by ELISA and IFA
Detection of antibodies to SVA VP1, VP2, and VP3 protein was performed by indirect ELISA as previously described [12, 13] on microtiter plates coated with either 500 ng or 200 ng of antigen per well or a combination of all 3 proteins at 100 ng each (300 ng total protein per well). Positive and negative control serum samples were run on each plate.
An avidity ELISA was performed following the ELISA protocol above, coating with 200 ng of VP2 antigen per well, and with the addition of a guanidine HCl wash step. Before secondary antibody was added, 1 M guanidine HCl in phosphate buffered saline (PBS) + 0.05% Tween-20, pH 7.4, was added to each well and incubated for 10 min. Plates were then washed as usual, secondary antibody was added, and the remainder of the ELISA protocol was performed as above. The avidity index was determined by dividing the optical density (OD) of the sample treated with guanidine by the OD of the sample without guanidine treatment (ODGn+/ODGn-).
The UMN-VDL performed a diagnostic IFA test to detect anti-SVA antibodies present in serum. Briefly, human lung cancer NCI-H1299 cells were inoculated with an SVA strain isolated in 2015 from an outbreak in the U.S. Infected cells were washed with PBS, fixed with acetone and incubated using two-fold dilutions of serum from 1:20 to 1:320 at 37 °C for 1 h. After fluorescein labeled goat anti-pig IgG diluted 1:50 in PBS was added into the wells and incubated at 37 °C for 1 h, the cells were observed under fluorescence microscopy. A positive signal at a sample dilution of 1:20 was considered suspect and a 1:40 or higher dilution was considered to be positive.
Statistical methods
ELISA analysis, receiver-operator characteristics (ROC) analysis, and comparison to the IFA results were performed using GraphPad Prism software (Version 5.0a, GraphPad Software, Inc., La Jolla, CA).