Inhibition of porcine reproductive and respiratory syndrome virus replication by RNA interference in MARC-145 cells

With the ultimate aim of producing an RNA interference-mediated transgenic pig that is resistant to porcine reproductive and respiratory syndrome virus (PRRSV), we have investigated the effect of RNA interference (RNAi) on silencing the expression of viral genes in the MARC-145 cell line. Twenty small interfering RNAs (siRNAs) were designed and screened for their ability to suppress the expression of the genes ORF1b, 5, 6, and 7 from the highly virulent isolate, PRRSV-JXwn06. Of these siRNAs, the four most effective were selected and four short hairpin RNA (shRNA) expression vectors (pGenesil-1-1b-135, pGenesil-1-1b-372, pGenesil-1-6-135, and pGenesil-1-6-169) targeting ORF1b and ORF6 were constructed and delivered into MARC-145 cells. These cells were then infected with JXwn06. All four vectors inhibited the PRRSV-specific cytopathic effect (CPE). The virus titers in cells transfected with pGenesil-1-1b-135, pGenesil-1-1b-372, pGenesil-1-6-135, and pGenesil-1-6-169 were lower than that of control cells by approximately 150-, 600-, 2.3- and 1.7-fold, respectively. In addition, the expression levels of ORF1 and ORF6 were reduced compared with controls. The unglycosylated membrane protein M, encoded by ORF6, was not detectable in cells transfected with shRNA expression vectors. These results verified that RNAi can effectively inhibit PRRSV-JXwn06 replication in cultured cells in vitro. The four shRNA expression vectors are an initial step in the production of transgenic pigs with PRRSV resistance.