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

Author: LvJianLiang
Tutor: ZhangYongGuang
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Foot and mouth disease virus Genome Infectious cDNA Cloning Chimeric virus Reverse genetics False nodules Internal ribosome into the sequence
CLC: S852.65
Type: Master's thesis
Year: 2008
Downloads: 169
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Abstract


FMD (Foot-and-Mouth Disease, FMD) is a serious threat to the world economy and human health, animal husbandry deadly infectious diseases, cattle, sheep, pigs and other major livestock number one killer. The disease pathogen FMDV (Foot-and-Mouth Disease virus, FMDV) are small RNA virus with a high degree of variability and a wide host range, pathogenicity on its molecular mechanism of the molecular mechanism of antigenic variation and host tropism, has is not very clear. In order to study its pathogenesis, molecular mechanisms of antigenic variation and host tropism, we constructed the first pan-Asian type O/CHINA/99 bovine FMDV strains three different infectious cDNA molecular cloning. 1. FMDV O/CHINA/99 strains of full-length cDNA construct molecular cloning and identification of infectious cover O/CHINA/99 designed and synthesized the genome of nine strains of primers from infected cattle O/CHINA/99 rat strains carcass extracted viral genomic RNA, using RT-PCR were amplified gene fragment of 4 entries (S, C, Z and E). Both ends of the fragments using restriction endonuclease sites, each of the gene fragments were inserted into the pOK12 vector to construct a strain genome O/CHINA/99 Molecular Cloning full-length cDNA. Liposome transfection method transcript RNA into BHK-21 cells, 24h after observing the typical FMDV cytopathic effect. Respectively, using RT-PCR method and suckling mice intradermal inoculation method rescued viruses were identified, the results show that salvation to a particular FMDV. These identification results show that we have successfully constructed O/CHINA/99 strains of full-length infectious cDNA molecular cloning and genetic engineering rescued virus. This will study FMDV genome structure and function of the molecular pathogenesis explore PMDV, and host tropism has laid a good foundation. 2. FMDV O/CHINA/99 deletion strains constructed full-length cDNA molecular cloning and identification of infectious functions on PKs currently not clear, there were reports that PKs with FMDV host tropism and virulence of a certain relationship . To reveal the FMDV translation, as well as to clarify the molecular pathogenesis of viral PKs gene deletion causes changes in host tropism of the reasons we designed and synthesized O/CHINA/99 strain genome five primers by RT-PCR amplification of gene fragments, 0/CHINA/99 deletion strain was successfully constructed full-length cDNA Molecular Cloning. And save a lot of missing out 0/CHINA/99 genetically engineered virus, which is to further explore the molecular mechanisms of pathogenesis FMDV and host tropism has laid a good foundation. 3 chimeric virus pOKTAW97/CHINA99 full-length cDNA construct molecular cloning and identification of infectious protein translation IRES is cis-acting elements, secondary structure has four domains, IRES cap structure does not depend guidance viral protein synthesis. Since changes in the spatial structure of IRES, and a combination of a host of different eukaryotic translation initiation factor, the virus IRES translation and host tropism play a very important role. To reveal the FMDV protein translation, as well as to clarify the molecular pathogenesis of IRES change in the spatial structure of the virus host tropism causes of change, we strain of bovine FMDV O/CHINA/99 infectious cDNA for the skeleton, with pig source of FMDV IRES replacement of the corresponding section, constructed FMDV type virus pOKTAW97/CHINA99 fitted into the full-length cDNA was cloned, and the obtained displacement of the chimeric virus IRES. This will study FMDV IRES genome structure and function, explore the molecular pathogenesis of FMDV, and host tropism has created good conditions.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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