Construction And Identification Of Infectious Molecular Clone Of Foot-and-Mouth Disease Virus (Full-Length,Deletion And Replacement) Strain O/CHINA/99

Foot-and-mouth disease, one of the most important disease of livestock, is caused by a small RNA virus of the family Picornavh'idae. FMDV showed high variability, host tropism and the mechanism of its molecular pathogenesis hasn't been clarified. In order to research high variability and host tropism,we sequenced the full length genome of FMDV O/CHINA/99 strain isolated from cattle, and generated an infectious cDNA clone of the strain.1. To construct the full-length cDNA of FMDV O/CHINA/99 strain, we design four pairs of over-lapping primers, which cover the whole genome of FMDV O/CHINA/99 strain, according to the nueleotide sequence of O/CHINA/99 strain. After extracting the total RNA of virus from the infected newborn mice, four cDNA fragments (S, C, Z and E) of O/CHINA/99 strain were amplified by reverse transcription PCR. These PCR products and pOK12 vector were digested with the unique restriction enzymes respectively, and ligated into pOK12 vector. Finally, the full-length cDNA of O/CHINA/99 strain was constructed. RNA synthesized in vitro by means of a T7 promoter inserted in front of the cDNA lead to the production of infectious particles upon transfection of BHK-2I cells, as shown by eytopathic effects.The rescued Virus was also found to be highly pathogenic for mice by intradormal injection producing afatal disease indistinguishable from that of wild-type virus. A full-length cDNA clone of a FMDV isolated from swine was assembled in the plasmid vector pOK12.2. FMDV change of the host tropic,lack strains most likely to cause the host tropic change. PKs on the function is still not clear, it is reported that the PKs with the FMDV host tropic and toxicity of a certain relationship. To reveal FMDV virus translation, molecular pathogenesis and clarify PKs gene deletion led to the virus host tropic the causes of change, we design and synthesis type O FMDV behalf of Pan-Asian strain O/CHINA/99 Genome five primer. The results showed that the full-length cDNA molecule of FMDV O/CHINA/99 strain was construeted successfully. The results showed that by the O/CHINA/99 deletion of genetic engineering virus, FMDV to further explore the molecular mechanism of pathogenesis and host tropic laid a good foundation.3. IRES is the translation of cis-element and have two of four Central domain. IRES guidance virus protein synthesis that don't rely on the structure hat. As IRES changes in the structure, and the combination of host different eukaryotic translation initiation factor, so IRES play an important role in the virus translation and host tropic.To reveal FMDV virus protein translation, molecular pathogenesis and clarify IRES spatial structure change in the host tropic virus the causes of change, we constructed a virus embedded FMDV pOKTAW97/CHINA99 the full-length cDNA molecular cloning and transcription by the virus RNA, using liposomes transfer method transcription of RNA into BHK-21 cells, by Replacement IRES the chimeric virus. This will study the IRES FMDV genome structure and function of PMDV the molecular pathogenesis and host tropic laid the foundation.