Abstract

To explore the application of RNAi technology for the control of duck hepatitis A virus type 1 (DHAV-1). The RNA-dependent RNA polymerase (RdRp) gene was amplified by PCR. pEGFP-RdRp was constructed for fusion expression of EGFP-RdRp protein. According to the sequence of RdRp, three gene-specific siRNAs were designed and the corresponding shRNA was inserted into pmiRZipΔ to construct pRdRp-shRNA. pRdRp-shRNA and pEGFP-RdRp were co-transfected into HEK293T cells for effective siRNA screening using fluorescent microscopy, flow cytometry, and quantitative fluorescence PCR. More effective siRNA was selected for further study using lentivirus vector pmiRZip. After demonstration of successful preparation of recombinant lentivirus, the suppressing effect was determined by calculating the 50% tissue culture infection dose (TCID₅₀) and RdRp gene expression of DHAV-1 in the duck embryo fibroblast (DEF) cells infected with recombinant lentivirus followed by DHAV-1 infection. The results indicated that all of the three siRNAs could suppress the RdRp gene expression, and the shRNA2 containing GDD motif had the highest efficiency. The recombinant lentivirus corresponding to shRNA2 reduced the TCID₅₀ of DHAV-1 by 6.2 l g and the RdRp gene expression by 89.6%, with the suppressing effect continued for at least 120 h. This work provides a new idea for the clinical control of duck virus hepatitis.

Key words: duck virus hepatitis, GDD motif, RNA-dependent RNA polymerase (RdRp), RNAi, siRNA.