*4.3. CSFV Ab B-ELISA and SN Tests*

Serum samples from wild boars were tested in a CSFV E2 Antibody ELISA. The CSFV Ab B-ELISA (BioNote Co. Cat. No. EB4413PO, Korea) is a competition ELISA designed to detect the E2 protein; however, it also provides a PI value (≥40% positive and <40% negative). Serum neutralization (SN) tests based on a neutralizing peroxidase-linked assay (NPLA) were performed to detect CSFV-specific neutralizing antibodies. Briefly, PK-15 cells inoculated with CSFV were incubated for 72 h, at 37 ◦C/5% CO2, with serum samples (serially diluted 2-fold). PK-15 cells were fixed in prechilled 80% acetone and then reacted with a 3B6 monoclonal antibody specific for CSFV E2 3B6 (Median Diagnostics Co., South Korea). PK-15 cells were then stained, using a VECTOR kit (Vector Laboratories, Burlingame, CA, USA), biotinylated anti-mouse IgG (H+L) (Cat. No. BA-9200), ABC solution (Cat. No. PK-4000), and a DAB peroxidase substrate (Cat. No. SK-4100). Staining was observed under a microscope.

## *4.4. Statistical Analysis*

All statistical analyses were performed by using GraphPad Prism software, version 6.0, for Windows. Data were analyzed by using two-way analysis of variance (ANOVA) with Bonferroni posttest.

**Author Contributions:** Conceptualization, S.C. and B.-K.P.; methodology, R.M.C., D.-S.Y., K.-S.K., S.S., B.-I.J., S.-H.C., and S.-I.L.; writing—review and editing, B.-H.H. and D.-J.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was supported by grants (Project Code No. B-1543083-2019-21-02) from the Animal and Plant Quarantine Agency and the Ministry of Agriculture Food and Rural Affairs, Republic of Korea.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Ethical Approval and Consent to Participate:** All experiments on hunted wild boars were approved by the Animal and Plant Quarantine Agency and the Ministry of Agriculture Food and Rural Affairs.
