Cultivation and Manipulation of Orf Virus

ORFV-GS14 preserved in our laboratory (an ORFV isolated from Gansu Province, China, designated as GS14) was used as a parent virus throughout the experiment and as a genetic background virus to generate target gene-deleted virus. Virus stocks were propagated in Primary Goat Testicular cells (PGT cells) and stored at −80 °C. The wild-type virus was prepared by sampling scabs from the goat and grinding them after the challenge experiment [3 (link)]. The cbp/gif double-gene deletion mutant (rGS14ΔCBPΔGIF) of ORFV was constructed in our laboratory [3 (link)]. The EGFP-N1 and other plasmids were kindly donated by the China Institute of Veterinary Drug Control. The PGT cells were prepared and frozen in our laboratory [3 (link),27 (link)]. Sheep testis cells were cultured in Ham’s DMEM/12 Gluta MAX medium (Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS) (Every Green, Zhejiang Tianhang Biotechnology Co., PGTd, Hangzhou, China) and 1% penicillin-streptomycin solution (with a final concentration of 100 IU/mL of penicillin and 100 µg/mL of streptomycin) at 37 °C with 5% CO₂.