Transgenic - Mediated RNA Interference In Resistance To Foot - And - Mouth Disease Virus Infection

Foot-and-mouth disease virus (FMDV) is responsible for the heavy economic losses across the world each year. Because of the deficiency of existing vaccines, the development of new antiviral strategies is needed. RN A interference (RNAi) is a recently found mechanism which induces gene silencing post-transcriptionally in all eukaryotes and a lot of investigative works have been performed on its potential of overcoming virus infection. However, current techniques have their own limitations of conducting RNAi persistently and effectively in vivo. In this study, we tried to endue cultured cells and mice with the ability of expressing anti-FMDV shRNAs endogenously by transgenic technology. Fisrt, two recombinant plasmids PB-EN3D2B and PB-N3D2B were constructed, both of which encode two shRNAs that target the 3D and 2B non-structural regions of FMDV genome. By employing piggyBac transposon system to integrate PB-EN3D2B into swine cell IBRS-2 or hamster cell BHK-21, we got 27 stable monoclonal transgenic cell lines.3 of 6 IBRS-2 transgenic cell lines exhibited innate resistance against both type O and type AsiaI FMDV:IB32-2 and IB32-3 displayed 10 to 1000 times of inhibition of the virus replication within 48 hours post challenge, when the cell line IB32-6 was intact in all three experiments without any CPEs. The results implied that this resistance was brought by endogenous shRNAs but not interferons and was not directly relevant to transgenic copy numbers. Then the 2.5kb shRNA-expressing fragment of PB-N3D2B was transfected into mouse fertilized eggs by microinjection or by lenti-virus vector to harvest 27 primary transgenic mice. Offspring of transgenic mouse strain F3D2B-10, N3D2B-18, and N3D2B-81 received type O FMDV challenge and the transgenic suckling mice of strain N3D2B-18 and N3D2B-81 showed 19 to 27.3 percentage of increase in surviving rates when injected with 3 to 6 LD50 of type O FMDV. But strain F3D2B-10 and N3D2B-18 failed to inhibit the replication of type AsiaI FMDV in subsequent experiments. This investigation was the first to detailedly study the feasibility and validity of introducing RNAi into mammals to overcome FMDV infection by transgenic strategy both in vitro and in vivo. We wish our work could provide useful information for similar applications on large susceptible animals like swine and bovine.