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Infectious bursal disease virus (IBDV) causes an important disease in young chickens. Chicken heat-shock protein 90 (cHsp90) has been shown to be a functional component of the cellular receptor complex for IBDV infection. This study demonstrates the inhibitory effect of vector-expressed anti-cHsp90α microRNA (miRNA) on IBDV infection. The reporter vectors pcHsp90α-EGFP and pcHsp90β-EGFP were constructed to facilitate effective miRNA selection. Two anti-cHsp90α and one anti-cHsp90β miRNA-expression vectors were constructed for a stable transfection study. Poly(A)-tailed RT-PCR detected sequence-specific miRNA transcription in transfected cells. Semiquantitative RT-PCR showed inhibition of cHsp90 transcription in transfected cells. A virus-titration assay showed that the anti-cHsp90α miRNA, but not the anti-cHsp90β miRNA, had inhibitory effects on IBDV infection. These results suggest that cHsp90α is a functional component of the cellular receptor complex for IBDV infection, and that anti-cHsp90α miRNA could be used as an anti-IBDV reagent.

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-RFPmiVP1 or 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.

RNA interference (RNAi) is a potent mechanism against a variety of viral infections. Infectious bursal disease virus (IBDV) causes an important disease economically in chickens, which is difficult to control. As part of the development of viral vector-mediated RNAi strategy against the disease, five anti-VP2 small interference RNAs were selected for construction of microRNA (miRNA) expression vectors tailored for avian cells. Transfection of DF-1 cells with the five vectors resulted in significant inhibition of VP2-EGFP reporter gene expression. More effective miVP2A and miVP2E were selected for further study using single or double miRNA expression vectors. After demonstration of specific miRNA expression, the gene silencing effects were determined in the vector-transfected and IBDV-infected cells. Reverse transcriptase PCR and virus titration showed inhibition rates from 76 to 82% on VP2 expression and significant decreases in virus titer by individual and co-expressed miVP2A and miVP2E. The inhibitory effects lasted for at least 120 h after infection with IBDV. These data suggest that the miRNAs targeting the VP2 can inhibit efficiently replication of IBDV., (Copyright 2010. Published by Elsevier B.V.)