*4.7. Animal Experiment*

The animal experiment was approved by the Research Ethics Committee of College of Veterinary Medicine, Huazhong Agricultural University, Hubei, China (No.20190526). Twenty 8-week-old three-breed cross pigs were purchased from the experimental farm of Huazhong Agricultural University and randomly divided into four groups. All pigs were confirmed to be seronegative for CSFV by neutralization test and RT-PCR. The four groups of pigs were housed in the negative-pressure facility of Wuhan Keqian Biology Co., Ltd (Wuhan, China) and were placed in separate rooms to avoid cross infection.

Pigs in group A (control group) were inoculated intranasally with 2 <sup>×</sup> <sup>10</sup><sup>6</sup> TCID50 of virulent CSFV SM strain. Pigs in group B were inoculated intranasally with 2 <sup>×</sup> <sup>10</sup><sup>6</sup> TCID50 of HBXY-2019 strain. Pigs in group C were inoculated intranasally with 2 <sup>×</sup> 106 TCID50 of GDGZ-2019 strain. Pigs in group D were inoculated intranasally with 2 <sup>×</sup> <sup>10</sup><sup>6</sup> TCID50 of GXGG-2019 strain. Following challenge, the rectal temperature and clinical signs (liveliness, body tension, body shape, breathing, neurological signs, conjunctivitis, appetite, defecation and so on) were monitored daily. A rectal temperature above 40.0 °C is considered an indication of fever. The clinical scoring (CS) system was used to evaluate the virulence

of CSFV in accordance with previously established standards [23]. Clinical scores were evaluated based on 10 clinical parameters [23]. Each parameter was calculated as follows: normal, 0 points; slightly altered, 1 point; distinct clinical signs, 2 points; severe CSF symptom, 3 points. The maximum total score per pig was 30.
