*4.3. Immunohistochemical Assay and qRT-PCR*

IHC staining was performed as described previously [23,24]. CSF antigens derived from the ND20, HY58, and GPE<sup>−</sup> strains were detected using DAB and the EnVisionTM peroxidase-conjugated polymer reagent (DAKO, Denmark). The results were scored according to the strength of straining as follows: +: 1–3 foci/section, ++: 4–10 foci/section, +++: >10 foci/section. The CSF RNA copy number within the organs of pigs was measured using quantitative real-time PCR (qRT-PCR) and calculated as log 10/g. The AnyQ CSFV qRT-PCR (Median Diagnostic Co. Cat No. NS-CSF-31, Korea) system and TaqMan probes targeting the 5'-UTR region with high specificity were used. The reaction conditions for qRT-PCR have been reported previously [24]. IHC staining was performed on the pigs showing most severe or mild clinical signs of each group. Whereas, qRT-PCR was performed on all pigs of each group.

**Author Contributions:** Conceptualization, S.C., V.P.L., and D.-J.A.; methodology, K.-S.K., J.-H.K., S.S., R.M.C., J.S., G.-N.P., and T.L.N.; writing/review and editing, B.-H.H., B.-K.P., 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. I-1543083-2019-20-01) from the Animal and Plant Quarantine Agency and 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 animal experiments were approved by Institutional Animal Cure and Use Committee (IACUC) of the Animal and Plant Quarantine Agency (APQA) in Ministry of Agriculture Food and Rural Affairs. All experiments were performed in secure biosafety level laboratories of APQA according to Chapter 1.1.4 biosafety and biosecurity guide in World Organization for Animal Health. 2018.
