Retroviral Vector Mediated Inhibition Of CSFV's Poliferation In PK-15 Cells By ShRNA

Classical swine fever (CSF) is a highly contagious disease of pigs, which leads to important economic losses worldwide. The etiological agent of CSF is classical swine fever virus (CSFV), which is a member of the genus Pestivirus within the family Flaviviridae. The current preventive and control measures of CSF epidemic are vaccine inoculation or slaughter the infected pigs. Although this has prevented the disease efficiently, failure of immunization and persistent infection of CSFV occur occasionally. Thus, the development of new antiviral methods is necessary in order to prevent the prevalence of CSF. RNA interference (RNAi) refers to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell. This phenomenon can result in highly specific suppression of gene expression, which is mediated by 21- to 23-nucleotide small interfering RNA (siRNA) that is homologous in sequence to the silenced gene. RNAi can shut down or regulates gene expression in mammalian cells and confers as a cellular defense mechanism against gene invaders such as transposons and viruses. SiRNA has showed its potential antiviral abilities in cultured mammalian cells and animals, and has been used as for gene-specific therapeutics for viral disease. Retroviral vectors offer is their ability to transform their genome into a double stranded DNA molecule that stably integrates into the target cell genome. Retroviral vector-mediated gene transfer became a powerful tool in scientific research and gene therapy. Here we use retroviral vector-mediated CSFV-specific shRNAs inhibition of CSFV's Poliferation in PK-15 cells.CSFV-specific-shRNAs, N1,N2,S10 and S11, which target on Npro, NS3 and NS4A of CSFV genome, were obtained. Recombinant plasmids , pLN-N1,pLN-N2,pLN-S10 and pLN-S11, were constrcted, which express CSFV-specific-shRNAs under promoter H1. Each recombinant plasmids co-transfent packaging cell lines GP2-293 with pVSV-G to produce replication-incompetent particles. Electronic microscope picture and viral titer determining certificate recombinant viruses, and the titer of recombinant viruses are 3.5×104cfu/mL (rMLV-N1),2×104cfu/mL(rMLV-N2),1×105cfu/mL(rMLV-S10),1×105 cfu/mL(rMLV-S11). Then each recombinant virus infects PK-15 cells followed screening by antibiotics G-418, and stable expressing CSFV-specific-shRNA cells were selected, which can inhibit of CSFV's Poliferation.The features of inhibition of CSFV's Poliferation by shRNAs were analyzed by using indirect immunofluorescent assay (IFA) and real-time PCR assay and virus titration assay to detect CSFV protein expression and virus genome RNA replication and assembly of matured virus particles, respectively. We nearly got the same results. The results showed that stable expressing CSFV-specific-shRNA cells effectively suppress CSFV's poliferation at 12-60h post-infection, and the cells can suppress CSFV's poliferation even at 120h post-infection. In PK-15-N2 cells, CSFV genome RNA replication level were 1/51.6,1/149.3 and 1/57.7 less than that of control at 24h,48h and 60h post-infection respectively. Matured virus particles level were 1/83.17,1/143.54 and 1/11.02 less than that of control at 24h,48h and 60h post-infection respectively. CSFV genome RNA replication level and Matured virus particles level were 1/9.2 and 1/16.78 less than that of control at 120h post-infection respectively. The results demonstrated that CSFV- specific-shRNAs inhibited the syntheses of virus protein, which lead to deficiency of replication of virus genome RNA, and assembly of virus particles, subsequently. Nest-PCR and continuous passage of stable expressing CSFV-specific-shRNA cells demonstrate that CSFV-specific-shRNAs stably integrates into PK-15 cell genome and inhibit of CSFV's poliferation.