*4.10. The 50% E*ff*ective Concentration (EC50) of Each Serum against Marker Virus*

For the calculation of EC50, the diagram of obtained luciferase activity was figured out as a sigmoid curve using ImageJ (version 1.52t) [42] Then, the EC50 was calculated based on the maximum and minimum value of each test.

#### *4.11. Ethics Statement*

The animal experiments were authorized by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, Hokkaido University (approval number 18-0038), and performed according to the guidelines of this committee.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-0817/9/3/188/s1: Figure S1: Serum neutralization test (SNT) using recombinant CSFV carrying HiBiT and anti-pestivirus serum; Figure S2: SNT using recombinant BVDV-1 carrying HiBiT and anti-pestivirus serum; Figure S3: SNT using recombinant BDV carrying HiBiT and anti-pestivirus serum, Table S1: Primers for the construction of HiBiT recombinant full-length cDNA of BDV and the virus rescue for BVDV-1 and BDV.

**Author Contributions:** Conceptualization, K.M. and Y.S.; methodology, T.T., T.F., N.T., and Y.S.; validation, K.M. and M.O.; formal analysis, M.T.; investigation, M.T. and T.K.; resources, Y.M. and Y.S.; data curation, M.T.; writing—original draft preparation, M.T.; writing—review and editing, K.M. and Y.S.; supervision, Y.S.; funding acquisition, K.M., M.O., and Y.S. All authors have read and agree to the published version of the manuscript.

**Funding:** This study was partially funded by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan (MEXT KAKENHI Grant Numbers: JP19H03115, JP16H06429, JP16K21723, and JP16H06431); and the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) (Grant Number: PJ18fm0108008) from the Japan Agency for Medical Research and Development (AMED).

**Acknowledgments:** Our great appreciation is extended to T. B. Rasmussen and C. Johnston, National Veterinary Institute, Technical University of Denmark, for the revision of constructing the BAC clone.

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

#### **References**


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