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The Infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family and the Aquabirnavirus genus, with salmonid fish as its primary host. Using vector-based shRNA molecules against the target genes has presented a new brand and effective therapeutic approach in using the RNAi pathway. Therefore, in the present study, the appropriate oligonucleotide sequence for encoding the gene of the generator gene of RNA polymerase of the IPNV virus was chosen, using the NCBI database. The designed shRNA structures were validated by online shRNA design software and further confirmed by blasting them against the fish genome in the NCBI database. Subsequently, shRNA oligonucleotides were synthesized and cloned into a lentiviral plasmid. Lentiviruses containing shRNA were produced in HEK 293T cells using a lentiviral packaging system. Following this, the lentivectors expressing shRNA were introduced into the CS2-2 cell line infected with the IPNV virus. The effectiveness of the designed shRNA molecules in suppressing the expression of the viral RNA polymerase gene was evaluated using the TCID50 test. The results indicated a significant reduction in viral titer, up to 99.9%, suggesting the potential of shRNA as a defense tool against IPNV virus in cell culture applications. This study highlights the promising role of shRNA technology in combating viral infections, explicitly targeting IPNV in this context.