Complete Genomic Sequencing And Construction Of An Infectious CDNA Clone Of A Cell Passaged FMDV OH/99 Strain Isolated From Infected Swine

Foot-and-Mouth Disease (FMD) is one of the most important disease of cloven-hoofed animals. It seriously influence development of the animal husbandry and cause considerable economic loss. Therefore,all the countries of the world pay much attention to its study and prevention. In this paper , several pairs of primers were designe for sequencing the genome of FMDV OH/99 strain, and the complete sequence of OH/99 was obtained by RT-PCR and 3′-RACE methods. The 3′-RACE fragment (about 7300bp) was inserted into the pGEM–T Easy vector, and S fragment of 5′-untranslated region (about 377bp) and internal ribosome entry site (about 750bp) sequence were separately amplified and cloned into pGEM–T Easy vector for sequencing. These two fragments were ligated together by ligation PCR and a 15bp length poly(C) fragment was inserted between them. These ligation products and 3′-RACE fragment were subcloned into pBluescript SK vector, a full–length cDNA clone of FMDV OH/99 strain has been constructed.RNA synthesized in vitro by means of a bacteriophage T7 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cell using lipofectamine reagent. The rescued virus have highly pathogeniesis after inoculation baby mice. The virus was further confirmed by RT-PCR and electron microscope observation. The results show an infectious cDNA clone of FMDV OH/99 strain was obtained. It will provide a very useful tool for FMDV molecular biology characteristics research and design new FMD vaccine.