Producing MARC-145 cell lines with shRNA-mediated silencing of the Tsg101 gene: Three g of pGFP-V-RS vector expressing a scrambled shRNA or a 29 nt shRNA (OriGene, Rockville, MD, USA) against a conservative region between human and simian TSG101 cDNA, was used to transfect MARC-145 cells growing in 6-well culture plates. Transfected cells were selected with 0.8 µg/mL of puromycin (Invitrogen) to obtain individual colonies. Approximately 30 puromycin-resistant colonies were picked and two of them showing significant suppression of TSG101 at both RNA and protein levels were subcultured for loss-of-function studies of the role of TSG101 in PRRSV infection. Primers used for RT-PCR detection of tsg101 were generated against consensus sequence of monkey, human and porcine tsg101 cDNAs (GenBank TM accession numbers, NM_001195481, NM_006292 and JN882576, respectively), which allowed us to detect both monkey and porcine tsg101 with the same pair of primers. The primers were 5′-ATACCCTCCCAATCCCAGTGGTTA-3′ (sense) and 5′-ATCCATYTCCTCCTTCATCCGCCA-3′ (antisense, Y = C or T). Anti-TSG101 monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). The involvement of TSG101 in PRRSV infection was evaluated by comparing the replication kinetics of PRRS viruses between cells with or without TSG101-suppression [47] .