Inhibition Of IBDV Replication By Plasmid Vector-or Recombinant Avian Adeno-associated Virus-Delivered MiRNAs

Infectious bursal disease virus(IBDV) is the causative agent of a highly contagious disease in young chickens known as infectious bursal disease(IBD).IBDV infections cause not only different degrees of mortality in chicks,but also vaccination failure to other diseases.Presently,IBD is controlled mainly by vaccination,but its protective effect is compromised by the apoptotic effect of live vaccines on the bursa of the vaccinated chickens and recent emergence of very virulent IBDV strains.Therefore, novel strategies are needed for effective control of the disease.RNA interference(RNAi) is a post-transcriptional gene silencing mechanism conserved in eukaryotes ranging from worms to humans,which has been shown to be a novel anti-viral strategy for a variety of viral infections.To investigate the feasibility of RNAi technology for suppressing IBDV infection,we in this study used the recently developed RNAi system tailored for chickens to drive miRNA expression.In both reporter vector-transfected and IBDV-infected cells,expressions of the two gene-specific miRNA resulted in significant but incomplete inhibition on VP2 gene expression and/or IBDV replication.These two miRNAs were then delivered by recombinant avian adeno-associated virus,showing significant inhibition on IBDV replication.These data demonstrate the highly effective inhibition of IBDV gene expression and viral replication by miRNAs targeting the VP2 gene.The more detailed experimental findings are summerized as follows.To investigate whether the human RNA polymerase H1 promoter can efficiently express small interference RNAs(siRNAs) in avian cells,10 siRNAs against green fluorescence protein(GFP) gene were predicted using the web-based siDirect software, one of which was selected for synthesis of short hairpin RNA(shRNA) by PCR.The shRNA was inserted into the RNAi vector pSuper containing the human H1 promoter, resulting in siRNA expression vector pSuper-shRNA.The H1-shRNA cassette was then subcloned into GFP gene expression vector pEGFP-H1,resulting in another expression vector pEGFP-H1-shRNA.The pEGFP-H1-shRNA or pSuper-shRNA+ pEGFP-N1 vector was transfected into simian COS-1 cells,human 293-T cells,chicken embryonic liver(CEL) cells and chicken embryonic fibroblast(CEF) cells and the transfected cells were submitted to fluorescence microscopy.Compared to pEGFP-N1-transfected cells, significant decreases in fluorescence density were evident in the mammalian cells transfected with pEGFP-H1-shRNA or pSuper-shRNA+ peGFP-N1 from 24 h after transfection,which was not seen in the avian cells transfected with the same vector(s). These data demonstrate that human H1 promoter can efficiently transcribe shRNA in the mammalian cells,but the transcription activity in the avian cells is relatively low, suggesting that the promoters of avian origin should be used for expression of shRNA in avian cells.To verify the miRNA expression system in avian cells using the avian U6 promoter -controlled pRFPRNAiC vector,10 siRNAs against GFP reporter gene were predicted using Genscript software,one ofwhich was selected for double-stranded oligonucleotide synthesis by PCR.The oligonucleotide was inserted into the miRNA expression vector, resulting in the recombinant vector pRFPRNAiGFP,pEGFP-N1 or pRFPRNAiGFP + pEGFP-N1 vector was transfeeted into COS-1 cells,293-T cells,CEL cells and CEF cells,respectively,and the transfected cells were submitted to fluorescence microscopy. The results showed that the total fluorescence of the pRFPRNAiGFP + pEGFP-N1-transfeeted cells was significantly lower than that of pEGFP-N1-transfected cells,especially in avian cells.These data demonstrate that avian U6 promoter can efficiently transcribe shRNAs in both mammalian and avian cells and the pRFPRNAiC vector is a suitable vector for miRNA expression in avian cells.To investigate the feasibility of inhibiting IBDV replication using the miRNA expression vector pRFPRNAiC,the VP2 gene without its stop codon was amplified from the total RNA extracted from IBDV Lukert strain-infected cells and fused to the N-terminal of GFP gene in pEGFP-N1 vector,resulting in a reporter vector pVP2-EGFP. Five potential miRNAs targeting the VP2 gene of IBDV were selected for double-stranded nucleotide synthesis by PCR and subcloned individually into the pRFPRNAiC vector,resulting in miRNA expression vectors pRFPRNAmiVP2A, pRFPRNAmiVP2B,pRFPRNAmiVP2C,pRFPRNAmiVP2D and pRFPRNAmiVP2E.Each of the five miRNA expression vectors was co-transfected into DF-1 cells with the reporter vector pVP2-EGFP and expression of the five miRNAs was demonstrated by Northern dot blotting.The transfected cells were first submitted to fluorescence microscopy,showing significant decreases in GFP-positive cell number.Flow cytometry showed significant decrease in total fluorescene ranging from 59.7%to 78.5%.The two more efficient miRNAs,miVP2A and miVP2E,were subcloned into a neo gene-containing RNAi vector individually or in combination and the resulting recombinant vectors were transfected individually into DF-1 cells.After selection with G418 for 1 week,the transfected cells were infected with IBDV Lukert strain,and gene silencing effects on the VP2 expression and viral replication were tested by semi-quantitative RT-PCR and virus titration assay,showing decreases in virus replication by 5-61gs of TCID₅₀.These results provide strong evidence that miRNAs targeting the VP2 gene can efficiently inhibit gene expression and/or replication of IBDV.To further investigate the inhibitory effect of the miRNAs delivered recombinant AAAV,the miVP2E and miVP1(targeting the VP1 gene) together with RFP expression cassette were subcloned individually into AAAV transfer vector pAITR.The resulting vectors were co-transfected individually into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper.The presence of the miRNA expression cassettes in the recombinant AAAV particles was shown by PCR.DF-1 cells in each of 35-mm dishes were transducted with 8×10⁸ rAAAV and then infected with 3 different IBDV isolates 48 h after transduction.Ninety six hours after infection, semi-quantitative RT-PCR showed an 85.2%(Lukert stain) decrease of the VP2 transcript in the miVPE-expressing cells,while an 89.6%decrease in the miVP1-expressing cells.Infectious virus titration showed an 61gs(Lukert stain),71gs (YEZ strain) or 2.51gs(LYG Strain) decrease of TCID₅₀ in the miVP2E-expressing cells and 71gs(Lukert stain),71gs(YEZ strain) or 6.51gs(LYG Strain) decrease in the miVP1-expressing cells.The inhibitory effect remained for at least 6 days after IBDV infection.