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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

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Abstract

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.

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Acknowledgements

This research was a part of the project titled “Development of Fish Vaccines and Human Resource Training,” funded by the Ministry of Oceans and Fisheries, Korea.

Funding

This work was funded by the Ministry of Oceans and Fisheries, Korea. The project title is “Development of Fish Vaccines and Human Resource Training.”

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Authors and Affiliations

  1. Department of Aquatic Life Medicine, Pukyong National University, Busan, 48513, South Korea

    Jun Soung Kwak & Ki Hong Kim

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  1. Jun Soung Kwak
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  2. Ki Hong Kim
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JSK carried out the experiment and wrote the original draft preparation. KHK supervised the study and did the writing-reviewing and editing.

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Correspondence to Ki Hong Kim.

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Key Points

• Self-inhibitory VHSV was rescued by inserting P gene targeting-microRNA into the genome.

• Dicer gene knockout EPC cells were established using CRISPR/Cas9.

• Replication of self-inhibitory VHSV was enhanced in EPC-ΔDicer cells.

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Kwak, J.S., Kim, K.H. 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. Mar Biotechnol 23, 546–559 (2021). https://doi.org/10.1007/s10126-021-10045-4

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