*4.7. Cross-Neutralizing Antibodies against Three Genotypes of CSFV*

The genotype-specific neutralizing antibodies were cross-neutralized with sera from pigs at 0 dpi (before inoculation) to 14 dpi (or the end of the experimental period) against CSFV strains of genotypes 1.1 (LPC/AHRI strain), 2.1 (TD/96 strain) and 3.4 (94.4 strain). Two-fold serial diluted 56 ◦C heat-inactivated sera were mixed with equal volumes of 100 TCID50 of the viruses, incubated at 37 ◦C for 1 h, and subsequently transferred to PK-15 cells in 96-well plates. The starting dilution of each serum was 1:4. At 72 hpi, the cells were fixed and stained for the presence CSFV antigen by the IFA. Neutralizing titer is the log2 of the antibody dilution factor (reciprocal of dilution) when 50% of the wells are protected from infection.

#### *4.8. Indirect Fluorescent Assay (IFA)*

The inoculated cells in 96-well plates were fixed with 10% formaldehyde at room temperature for 10 min and washed three times with PBS. Each mAb against CSFV was diluted 1:100 in PBS, and 50 μL of the diluted mAb was added per well (mAb T6 for virus titration of the TD/96 strain and mAb L71 for virus titration of the 94.4 strain; mAb WH303 for detection of cross-neutralizing antibodies). The cells were then incubated at 37 ◦C for 1 h and washed three times with PBS. Fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1:100 in PBS was added, 50 μL per well. The cells were incubated at 37 ◦C for 1 h and then washed three times with PBS. Fluorescence of the stained cells was observed under a fluorescence microscope (Olympus Imaging America, Center Valley, PA, USA).

## *4.9. Statistical Analysis*

Differences in the values between two groups and among various groups were statistically analyzed using the Student's t-test and one-way analysis of variance (ANOVA), respectively. ANOVA was combined with the Duncan multiple range test. The statistical analysis was carried out in Statistical Analysis System (SAS) Enterprise Guide 7.1 (SAS Institute Inc., Cary, NC, USA). Mean differences were considered statistically significant when the *p*-value was <0.05.

**Author Contributions:** Conceptualization, Y.-L.H., C.-C.H., and C.-Y.C.; Methodology, Y.-L.H., H.-M.L., and C.-Y.C.; Validation, Y.-L.H. and C.-Y.C.; Formal Analysis, Y.-L.H. and H.-M.L.; Investigation, Y.-L.H., K.-J.T., M.-C.D., H.-M.L., and C.-Y.C.; Resources, Y.-L.H., K.-J.T., M.-C.D., and C.-Y.C.; Data Curation, K.-J.T., M.-C.D., and F.-I.W.; Writing—Original Draft Preparation, C.-Y.C.; Writing—Review & Editing, F.-I.W.; Supervision, F.-I.W.; Project Administration, C.-Y.C.; Funding Acquisition, C.-Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science and Technology [grant MOST 103-2321-B-062-001].

**Acknowledgments:** We particularly thank Yi-Hsieng Samuel Wu, Department of Animal Science and Technology, National Taiwan University, for his assistance in the statistical analysis. We thank Fan Lee, Animal Health Research Institute, for his critical comments on the manuscript.

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