Inhibition of infectious bursal disease virus replication in chicken embryos by miRNAs delivered by recombinant avian adeno-associated viral vector

Abstract:

Abstract: [Objective] We studied the inhibition of infectious bursal disease virus (IBDV) replication in chicken embryos by recombinant avian adeno-associated virus (AAAV)-delivered VP1- and VP2-specific microRNAs (miRNAs). [Methods and Results] We co-transfected AAV-293 cells with the VP1- or VP2 gene-specific miRNA expression vector pAITR-RFPmiVP1 or AITR-RFPmiVP2E, AAAV packaging vector pcDNA-ARC and adenovirus helper vector pHelper, resulting in recombinant virus rAAAV-RFPmiVP1or rAAAV-RFPmiVP2E. We also generated the recombinant viruses rAAAV-RFP (without miRNA expression cassette) and rAAAV-RFPmiVP2con (expressing control miRNA) using the same method as the control purpose. Electron microscopy showed that the recombinant viruses had a typical morphology of AAV. We confirmed the presence of miRNA expression cassette in the recombinant viral genomes by using PCR. Our poly(A)-tailed RT-PCR showed correct expression of the miRNAs in the rAAAV-transduced DF-1 cells. We inoculated the recombinant viruses individually into 8-day-old SPF chicken embryos and then challenged them using Lukert strain IBDV on day 2 after inoculation. Our IBDV titration assay showed that the 50% tissue culture infectious dose (TCID50) of rAAAV-RFP- or rAAAV-RFPmiVP2con-inoculated group was 8.0 log10, whereas the TCID50 of rAAAV-RFPmiVP1-inoculated group decreased to 1.0 and 0.8 log10 on day 3 and 6 after challenge, respectively. Similarly, the TCID50 of rAAAV-RFPmiVP2E-inoculated group decreased to 1.5 and 2.0 log10, respectively. [Conclusion] These data suggest that rAAAV can transduce efficiently chicken embryos and the expressed VP1- and VP2-specific miRNAs can inhibit the replication of IBDV efficiently.