RESUMO
Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.
Assuntos
Animais , Embrião de Galinha , Galinhas , Regulação para Baixo , Fibroblastos , Virologia , Marcação de Genes , Lentivirus , Genética , Metabolismo , Doença de Newcastle , Virologia , Vírus da Doença de Newcastle , Genética , Fisiologia , Fosfoproteínas , Genética , Metabolismo , Doenças das Aves Domésticas , Virologia , Interferência de RNA , RNA Interferente Pequeno , Genética , Metabolismo , Proteínas Virais , Genética , Metabolismo , Replicação ViralRESUMO
Fourteen H9N2 avian influenza viruses (AIV) were isolated from sick chickens in China from 1998 to 2008. The sequences of the Non-structural(NS) gene of these isolates were determined by RT-PCR and sequencing, and the entire ORF sequences of NS1 and NS2 protein were obtained.-The homology of these nucleotide sequences and the putative amino acid sequences were compared with several classic reference viruses of H9N2. These isolates were proved to be highly homologous in NS gene (92.9%-99.9% identity) and all belonged to A/Chicken/Beijing/1/1994-like group in the Asia bird-swine branch of allele A of HS gene phylogenetic tree.-According to this study and previous reports of other researchers, NS gene of H9N2 subtype AIV in chickens of China is genetically stable and there is no enough evidence to support the establishment of other sub-lineages in chickens.