Designing shRNA molecules targeting UL-25 gene as a gene therapy tool against BHV-1 virus

Bovine herpesvirus-1 (BHV-1) belong to the genus Varicellovirus in the subfamily Alphaherpesvirinae and the family Herpesviridae, the main host of the virus is cattle but Other Artiodactyla (goat, sheep and camel) may be infected. The BHV-1 cause the respiratory disease (IBR), with fever and abortion. Although the infection is infrequently life threatening, the introduction of cattle with BHV-1 can cause severe economic losses due to production losses and restrictions in the international trade of livestock.In recent years, RNAi has become a novel and effective tool to target gene silencing for functional genomic studies and antiviral therapy, The expression of shRNAs against target genes has now become a standard and powerful technology for gene silencing.The purpose of this study was design, synthesis and colon three shRNA molecules targeting UL-25 gene to inhibit virus replication.Several shRNA molecules was design by using some online software. Then three shRNA with appropriate size and situations were chosen, single strand oligonucleotide were synthesized and annealed with each other, eventually they were coloned in pCDH-CMV-MCS-EF1-cGFP-T2A-Puro viral plasmid vector.Due to verify accuracy and validity of cloning, colony-PCR and sequencing was performed.Consequently; To investigate the antiviral effects of shRNAs on BHV-1 replication, transfection of these viral plasmid vector in HEK 239T cell line and challenge with BHV-1 that inoculated in MDBK cell line will done.

Keywords:

shRNA , BHV-1 virus , UL-25 gene , gene silencing