Establishment And Evaluation Of Stable Cell Lines Inhibiting Foot-and-mouth Disease Virus By RNA Interference

Foot-and-mouth disease (FMD) is a kind of acute and highly contagious disease, which is caused byFoot-and-mouth disease virus (FMDV) and always be epidemic among cloven-hoofed animals such asswine, cattle and sheep. The epidemiological feature of FMD is characterized by a strong infectivity, arapid prevalence and a diverse of susceptible animal species, besides, FMDV has many serotypes andsubtypes, and the variants can be easily generated, bringing enormous difficulty in preventing. Currently,the main control strategy is vaccination and stamping out policy, which results in a high cost inpreventing and controlling. As the knowledge of the genome structure and function of FMDV has beenlargely enriched as well as the continuous development of the modern molecular biology techniques,some emerging technologies, such as antisense technology, RNA interference technology and transgenicbreeding for disease resistance, have been successfully employed in anti-FMDV research, which hasopened up a new route to prevent and control FMD.In present study, RNA interference technology was employed to screen effective RNA interferencetargets against the3D gene of FMDV genome. Firstly, to screen more effective candidate targetingregion in3D region of FMDV genome, nineteen complete genome sequences of serotype O FMDVwere downloaded from the GenBank database and the comparatively analysis of sequence wereimplemented by bioinformatics software MEGA4.1. Five potential targeting sites were predicted bysmall interference RNA (siRNA) designing software online. The transcriptional templates in vivo of thefive siRNA were synthesized and then cloned into the plasmid vector pSilencer4.1-CMV puro. Therecombinant plasmids were named p3D1shRNA, p3D2shRNA, p3D3shRNA, p3D4shRNA andp3D5shRNA, respectively. At the same time， blank control （BC） and negative control wereimplemented。Secondly, the recombinant plasmids above were transfected to baby hamster kidney cell lines(BHK21) by using of Lipofection2000Reagent. The cells after transfection were cultured in selectivemedium supplemented with puromycin (Puro,4μg/mL) for approximately14days to eliminate the cellsnot transfected, and then the macroscopic clones were picked out and continuously passaged in themedium supplemented with2μg/mL of puromycin. PCR assay was employed to determine whether thetransfected cells contain the recombinant vectors at the second, fifth, tenth, twentieth and thirtiethpassages. The results demonstrated that the recombinant vectors were successfully introduced into thecells and replicated stably following the passages of the transfected cells.Lastly, in order to evaluate the antiviral effect of the recombinant vectors in vivo, indirectimmunofluorescent assay, virus titration and quantitative real-time reverse transcription polymerasechain reaction were conducted, which represented protein expression level, progeny virions and nucleicacid replication level. After challenged with FMDV of O/CHA/99strain, two cell lines (p3D1shRNAand p3D4shRNA) showed a significant reduction in the synthesis of viral protein and RNA, as well as asharp decrease in virus yield, and the inhibition can last for at least thirty passages. We herein present aconvenient and efficient procedure for establishment and evaluation of stable cell lines for anti-FMDV research by RNAi, which can be as a reference for RNAi targets selection and as a cell model for longterm study.