Marc 145

PK-15, MARC-145, 293T, and COS-7 cells were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). MARC-145 and 293T cells were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum FBS (Gibco, Carlsbad, CA, USA). PK-15 and COS-7 cells were grown in minimal essential medium (MEM) supplemented with 10% FBS. PAMs were collected from PRRSV negative pigs (28 days old) and maintained in RPMI-1640 medium supplemented with 10% FBS. Wild-type highly pathogenic PRRSV (HP-PRRSV) SD16 strain was isolated by plaque purification and grown in MARC-145 cells as previously described53 (link). HP-PRRSV SD16-EGFP recombinant virus was engineered as described previously33 (link). Pig rotavirus (AV-55) was purchased from China Center of Veterinary Culture Collection and was propagated on MA-104 cells54 .

A mouse alveolar macrophage-derived cell line MH-S, a peritoneal macrophage-like cell line RAW264.7 and MARC-145 cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China). Primary PAMs were prepared from bronchoalveolar lavage of 4 to 6-week-old PRRSV-negative piglets. Culture and preparation of PAMs were conducted as previously described [32 (link), 33 (link)]. PAMs and the MH-S cell line were maintained in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (v/v; BI, Israel). RAW264.7 and MARC-145 cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) (BI).
Various genotype 2 PRRSV isolates including highly pathogenic PRRSV strains (listed with Genbank accession numbers in parentheses), JXA1 (GenBank: EF112445.1), SD16 (GenBank: JX087437.1), GD-HD (GenBank: KP793736.1) and classical strain VR-2332 (GenBank: AY150564 ) were used to infect the various cell lines at 0.1 to 10 multiplicity of infection (MOI). Viral titers were determined in MARC-145 cells by calculating the median tissue culture infective dose (TCID₅₀) as previously described [34 (link)].