To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P. The replication of rVHSV-A-amiR-C in EPC-ΔDicer cells was not different from that in control EPC cells, while the copy number of rVHSV-A-amiR-P was increasingly risen up in EPC-ΔDicer cells compared to that in control EPC cells, and the final viral titer of rVHSV-A-amiR-P was enhanced by culture in EPC-ΔDicer cells. These results indicate that VHSV can be attenuated by the equipment of self-mRNA-targeting microRNA sequence in the genome, and the titer of artificial miRNA-expressing attenuated recombinant VHSVs can be enhanced by the knockout of Dicer gene in EPC cells.

为了生产人工微RNA（amiR）介导的自我抑制病毒性出血性败血症病毒（VHSV），我们在VHSV的N和P基因之间插入了靶向VHSV P基因的amiR序列（amiR-P）或控制amiR序列（amiR-C）基因组，并使用反向遗传技术拯救了重组 VHSV（rVHSV-A-amiR-P 和 rVHSV-A-amiR-C）。与对照病毒 rVHSV-A-amiR-C 相比，rVHSV-A-amiR-P 的生长显着延迟，这是由于在受感染细胞中产生了攻击自身 P 基因转录物的 microRNA。为了增强 rVHSV-A-amiR-P 的复制，我们使用 CRISPR/Cas9 系统生成了 Dicer 基因敲除上皮瘤 papulosum cyprini (EPC-ΔDicer) 细胞，并评估了 Dicer 敲除对 rVHSV-A 滴度的影响-amiR-P。rVHSV-A-amiR-C在EPC-ΔDicer细胞中的复制与对照EPC细胞中的复制没有差异，而与EPC-ΔDicer细胞相比，rVHSV-A-amiR-P的拷贝数在EPC-ΔDicer细胞中逐渐升高。对照 EPC 细胞，在 EPC-ΔDicer 细胞中培养可提高 rVHSV-A-amiR-P 的最终病毒滴度。这些结果表明VHSV可以通过基因组中自身mRNA靶向microRNA序列的设备减毒，并且通过敲除EPC细胞中的Dicer基因可以增强表达人工miRNA的减毒重组VHSV的效价。