Inhibition Of Porcine Reproductive And Respiratory Syndrome Virus Replication By Short Hairpin RNA In Marc-145 Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, a disease characterized by reproductive failure in sows, including early farrowing with stillborn piglets and late-term abortions, as well as respiratory distress in young pigs and an influenza-like disease in grow-finish swine. Since it was first identified in Europe and United States in early 1990s, the disease is now endemic in many swine-producing countries and is one of the most economically important swine diseases. Current vaccines against PRRSV generally provide at best partial protection against clinical disease but do not prevent infection. Thus, development of new rapid-acting antiviral strategies effective against all virus isolates is imperative.RNA interference (RNAi) is a post-transcriptional mechanism of sequence-specific gene silencing that initiated by double-stranded RNA (dsRNA). By introduction of approximately 21 nucleotides (nt) duplexes of RNA(siRNA) or by transcription of DNA precursors into short hairpin RNAs (shRNAs) homologous to target sequences, the endogenous or pathogen mRNA can be inhibited. In current years, this technique has been used to attenuate viral infection in cell culture or animal experiments, which including hepatitis C virus (HCV), foot-and-mouth disease virus, influenza virus, hepatitis B virus (HBV). It has been raised expections about the use of RNAi as a new antiviral strategy,but little is known about RNAi on PRRSV.RNAi may provide a novel route to control virus since RNAi can inhibit gene expression high-efficiently and specificly. In this study, ability of pSUPER to initiate RNA interference was analysized. A recombinant plasmid with a short hairpin RNA (shRNA) targeting at the nuclear protein gene of influenza virus was constructed and identified by enzyme digestion and sequence analysis. The Hemagglutination(HA) titre and TCID₅₀ of virus in chicken embryo fibroblastic(CEF) cells transfected with the plasmid was lower than that of nontransfected with plasmid. It showed that the plasmid could specificly inhibit virus production in CEF cells.