Bovine fetal epithelium cells expressing shRNA targeting viral VP1 gene resisted against foot-and-mouth disease virus

RNAi could protect experimental animals from Foot-and-mouth disease virus (FMDV), but pivotal issue is delivery of RNAi. In this study, shRNA recombinant lentiviral plasmid RNAi-LT6 targeting VP1 of FMDV, which strongly suppressed the transient expression of a FLAG-tagged VP1 protein in 293T cells and significantly inhibited viral replication in BHK-21 cells, was screened and transfected into bovine fetal fibroblast cells. With subsequent somatic cell cloning, three 4-month-old transgenic fetuses were obtained, and integration into chromosome and expression of shRNA in primary transgenic tongue epithelium cells were confirmed by Southern hybridization and Northern assay, respectively. shRNA significantly suppressed viral RNA synthesis and viral replication in primary transgenic tongue epithelium cells after inoculation of 200 TCID50 of FMDV. The results suggested that transgenic cloning may be a useful tool for RNAi delivery and RNAi anti-viral strategy, and RNAi-LT6 could be a candidate shRNA used for preparation of transgenic cattle against FMDV.

► RNAi-LT6 against VP1 of FMDV strongly suppressed expression of VP1 in 293T cells. ► RNAi-LT6 significantly inhibited replication of FMDV in BHK-21 cells. ► 4-Month-old transgenic fetuses were obtained with subsequent somatic cell cloning. ► shRNA expression in primary transgenic fetal tongue epithelium cells was confirmed. ► shRNA strongly blocked viral replication in primary transgenic epithelium cells.