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Example:10.1021/acsami.1c06204 or Chem. Rev., 2007, 107, 2411-2502
Generation of Self-Inhibitory Recombinant Viral Hemorrhagic Septicemia Virus (VHSV) by Insertion of Viral P Gene-Targeting Artificial MicroRNA into Viral Genome and Effect of Dicer Gene Knockout on the Recombinant VHSV Replication
Marine Biotechnology  (IF3.619),  Pub Date : 2021-07-15, DOI: 10.1007/s10126-021-10045-4
Jun Soung Kwak, Ki Hong Kim

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.