RNA Interference Inhibits Replication Of Porcine Reproductive And Respiratory Syndrome Virus

N gene coding nucleocapsid (N) protein of PRRSV VR2332 resp strain was amplified by PCR, and cloned in the green fluorescence vector pEGFP-N1. Vector named as "PEGFP-N1-ORF7" with PRRSV VR2332 N gene was obtained by kanamycin screening. According to a Nature paper in 2004 and the RNAi targeting sequence software offered by MIT and Ambion website, two targeting sequences on N gene were choosed and blasted in order to show the low homologous to other species. Six cDNA strands were synthesized and annealed to form three double strand cDNAs, then cloned into the RNAi vector "pBS/U6 1.0". Validation of the RNAi vectors was done by the absent enzyme "Xhol" site. Finally, two RNAi vectors "pBS/U6-60", "pBS/U6-260" and a negative control "pBS/U6-Neg"were obtained.16 hours after co-transfection of RNAi vectors and the control vector with pEGFP-N1-ORF7, dramatically reduction in GFP amount compare to the control was observed. Western-blot and RT-PCR was performed to prove the specificity of the RNAi in this study. Results of western-blot showed that the inhibition percentage of pBS/U6-60 and pBS/U6-260 toward PRRSV N gene could separately reach at 82.90% and 69.95%. The conclusion was that RNAi specific to the two target sequeces could inhibit the PRRSV N gene expression in cells, and key sites for PRRSV replication maybe involved in these two target sequences.pEGFP-N1 vector was transfected in the MARC-145 cell lines to study the high transfection efficiency of RNAi vectors. When inoculation was performed at different times and at different TCID50(tissue culture infective doses), RNAi vectors and control vector were transfected in the MARC-145 cells and dramatically reduction of PRRSV replication by RNAi vectors was observed. In this study, results of TCID50 at different time, the occurrence time of CPE and IFA results in each group give the evidence that the antiviral effects could extend almost from 72 to 120 hours and could be useful in PRRSV prevention and therapy. Western-blot result of pBS/U6-60 group showed that, at 72 hours after inoculation, the amount of PRRSV N protein was reduced by 70.15% compare to the virus control. In conclusion, (I) RNAi could be used to inhibit PRRSV replication on MARC-145 cell lines;(II) Key sites for PRRSV replication maybe located in the two targeting sequences; (III) Our findings show that Vector-based RNAi technology would give a new vaccine design to PRRSV therapy.