Production and titration of recombinant PRRS viruses with expression of exogenous genes: In brief, two restriction enzyme digestion sites (Afl II and Mlu I) were introduced into 5'-and 3'-ends of the coding regions, which were amplified from authentic cDNA clones using a high-fidelity PCR [15, 47] . The cloning primers used for this study are listed in the Supplemental Table 1 . The amplified coding regions were purified and cloned into the expression cassette. MARC-145 cells were transfected with authentic plasmids purified from E. coli clones for examination of the production of progeny viruses and expression of indicator proteins or IFNs. Transfection of MARC-145 in 24-well plates was performed with 2 μg of plasmid DNA using Lipofectamine™ 2000 (Invitrogen). Subsequent virus yields were measured by end-point titration of culture media on MARC-145 cells and PAMs. Serial 10-fold dilutions of virus were placed in 96-well tissue culture plates containing confluent MARC-145 cells or PAMs. Cells were fixed in 4% formaldehyde in PBS and the virus was detected by staining for the presence of nucleocapsid antigen using a mAb (SDOW-17, Rural Technologies, Brookings, SD, USA) labeled with TRITC-conjugated secondary antibodies. In addition, the recombinant viruses could be distinctly detected by the expression of fluorescent proteins. Results were reported as log TCID50/mL [15, 47] . The replication of Rluc-expressing PRRSV was monitored with the addition of an in vivo Renilla luciferase substrate (Promega, Madison, WI, USA) at 60 μM in cell culture medium and measured the luminescence after 2 h [49] .