Cell Culture and PRRSV Propagation

Mouse fibroblast cell line L-929 (ATCC CCL-1) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient Mixture F-12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% non-essential amino acids. Pig kidney cell line PK-15 (ATCC CCL-33), mouse embryo fibroblast cell line NIH 3 T3 (ATCC CRL-1658), AAV-293 cells (Stratagene, USA) and African green monkey kidney cell line MARC-145 cells (ATCC CRL-12231) were grown in DMEM supplemented with 10% FBS and 1% non-essential amino acids. Primary PAMs were prepared from 6-week-old Lancrace pigs as described previously [7 (link)] and grown in DMEM/F-12 supplemented with 10% FBS, 1% non-essential amino acids and 20% L-929-conditioned medium. PRRSV strain VR-2332 (ATCC VR2332) is a prototype strain of North American genotype [28 (link)]. PRRSV strain CH-1R is an attenuated vaccine strain derived from the traditional Chinese strain CH-1a [29 ]. PRRSV strain JX-A1 is a highly pathogenic Chinese strain [30 (link)]. The three PRRSV strains were propagated and titrated on MARC-145 cells.