Wang Yongjuan, Sun Huaichang, Shen Pengpeng, Zhang Xinyu, Xia Xiaoli
College of Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, PR China.
J Gen Virol. 2009 Jun;90(Pt 6):1417-1422. doi: 10.1099/vir.0.010520-0. Epub 2009 Mar 4.
RNA interference (RNAi) is a novel antiviral strategy against a variety of virus infections. Infectious bursal disease virus (IBDV) causes an economically important disease in young chickens. This study demonstrated efficient inhibition of IBDV replication by recombinant avian adeno-associated virus (rAAAV)-delivered anti-VP1 and anti-VP2 microRNAs (miRNAs). In the viral vector-transduced cells, sequence-specific miRNA expression was detected by poly(A)-tailed RT-PCR. Reporter assays using a pVP2-EGFP vector showed significant and long-lasting inhibition of VP2-EGFP expression in cells transduced with anti-VP2 miRNA-expressing rAAAV-RFPmiVP2E, but not with the control miRNA-expressing rAAAV-RFPmiVP2con or anti-VP1 miRNA-expressing rAAAV-RFPmiVP1. Semi-quantitative RT-PCR and/or virus titration assays showed a significant inhibitory effect on homologous IBDV replication in cells transduced with rAAAV-RFPmiVP1 or rAAAV-RFPmiVP2E. For two heterologous IBDV isolates, transduction with rAAAV-RFPmiVP1 led to slightly weaker but similar inhibitory effects, whereas transduction with rAAAV-RFPmiVP2E resulted in significantly weaker and different inhibitory effects. These results suggest that rAAAV could act as an efficient vector for miRNA delivery into avian cells and that VP1 is the more suitable target for interfering with IBDV replication using RNAi technology.
RNA干扰（RNAi）是一种针对多种病毒感染的新型抗病毒策略。传染性法氏囊病病毒（IBDV）在雏鸡中引发一种具有重要经济影响的疾病。本研究证明了重组禽腺相关病毒（rAAAV）递送的抗VP1和抗VP2微小RNA（miRNA）可有效抑制IBDV复制。在病毒载体转导的细胞中，通过聚腺苷酸化尾RT-PCR检测到序列特异性miRNA表达。使用pVP2-EGFP载体的报告基因检测显示，在用表达抗VP2 miRNA的rAAAV-RFPmiVP2E转导的细胞中，VP2-EGFP表达受到显著且持久的抑制，但在用表达对照miRNA的rAAAV-RFPmiVP2con或表达抗VP1 miRNA的rAAAV-RFPmiVP1转导的细胞中未观察到这种抑制。半定量RT-PCR和/或病毒滴定分析表明，rAAAV-RFPmiVP1或rAAAV-RFPmiVP2E转导的细胞对同源IBDV复制具有显著的抑制作用。对于两种异源IBDV分离株，rAAAV-RFPmiVP1转导导致的抑制作用稍弱但相似，而rAAAV-RFPmiVP2E转导导致的抑制作用明显较弱且不同。这些结果表明，rAAAV可作为将miRNA递送至禽细胞的有效载体，并且VP1是使用RNAi技术干扰IBDV复制更合适的靶点。
