重组禽腺联病毒介导的miRNA抑制传染性法氏囊病病毒在鸡胚内的复制

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

摘要:

摘要：【目的】 在鸡胚水平上探索VP1和VP2基因特异miRNA抑制传染性法氏囊病病毒（infectious bursal disease virus, IBDV）复制的可行性。【方法与结果】将表达VP1基因特异miRNA重组载体pAITR-RFPmiVP1或VP2基因特异miRNA重组载体pAITR-RFPmiVP2E与禽腺联病毒（avian adeno-associated virus, AAAV）包装载体pcDNA-ARC和腺病毒辅助载体pHelper共转染AAV-293细胞，获得重组病毒rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E，用同样方法获得不表达miRNA的rAAAV-RFP和表达对照miRNA的rAAAV-RFPmiVP2con。电镜观察显示重组病毒具有典型的AAAV颗粒形态；PCR检测结果表明其基因组中含miRNA表达盒；经poly(A)加尾RT-PCR检测证明重组病毒感染细胞能表达基因特异的miRNA。分别将重组病毒经卵黄囊途径接种8日龄SPF鸡胚，然后经绒毛尿囊膜途径用Lukert株IBDV攻毒, 收获鸡胚进行IBDV组织细胞半数感染剂量（TCID50）测定。结果在攻毒后第3天，rAAAV-RFP和rAAAV-RFPmiVP2con接种组的IBDV TCID50为8.0 log10, rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E接种组的IBDV TCID50分别下降到1.0和1.5 log10；在攻毒后第6天，rAAAV-RFP和rAAAV-RFPmiVP2con接种组的IBDV TCID50仍为8.0 log10, rAAAV-RFPmiVP1和rAAAV-RFPmiVP2E接种组的TCID50分别下降到0.8和2.0 log10。【结论】 rAAAV是有效的miRNA鸡胚导入载体，表达的VP1和VP2基因特异miRNA能有效阻断IBDV复制。

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.