The Construction Of Infection Full-Length CDNA Of Asia1/WX Strain Of Foot-and-Mouth Disease Virus And In Vitro Rescue Of The Recombinant Virus

Foot-and-mouth disease(FMD), caused by Foot-and-mouth disesse viruse (FMDV), is an acute, heat resistance, highly contagious and systemic disease of domestic and wild cloven-hooved animal species. The viruse is continuous evolution within the natural process and occurs as seven distinct serotypes(serotypes O, A, C, SAT1-3and Asia1) and multiple subtypes, which make the viruse show a variety of phenotypic varuation within the host tropism, prospective species range, virulence, antigenicity and other aspects. In China, Asia1type FMDV mainly includes Yunnan58lineages, Xinjiang03lineages and Jiangsu05lineages, which later became the dominant linesge and prevalent strains. In2000, serotypes Asia1FMDV strain was isolated in Xinjiang Uyfur Autonomous Region of China and its representative strains is AKT/03, which mainly caused disease within cattle, but never within pigs. In2005, a new stain of serotypes Asia1was isolated in Jiangsu Wuxi region of The China, which caused the rapid spread of this stain, the researchers found that Jiangsu05lineages could infect swine and started to pay attention to this abnormal phenomenon. Through sequencing the pandemic strain within Jiangsu05lineages and aligning theses type Asia1FMDV strains sequence, this experiment objective is to show the differences of type Asia1FMDV strain isolated from swine and bovine in molecular genetics, and lay the foundation to study the pathogenic mechanisms and molecular variation mechanism of type Asia1FMDV from different sources.The reverse genetics techniques create a tool to generate the infection cDNA molecules for the reseach on foot-and-mouth virus, which can be used as a platform of virus genetic manipulation, providing the opportunity to further study the FMDV life cycle, however, the construct of FMDV full-length infection cDNA has become critical. To obtain the full-length infection cDNA, we used the Oligod(T)18as the reverse transcription primer and the RNA of type Asia1FMDV of Jiangsu05lineages as the template in the experiment to reverse transcript and obtained first strand cDNA, and later the fragment (about7500bp) covering the virus genome3’end full-length was amplified; with the specific reverse primer to reverse transcript, two small fragments locating around ploy(C) region in the5’UTR was obtained by PCR and the fragment about710bp was over-lap PCR amplified with the two short segments. With the restriction enzyme reaction, these amplified fragment were sequentially connected to the plasmid pSL1180, and then the full-length cDNA was constructed. In-vitro RNA transcript was transfected BHK21cells with liposomes and continuous passaged, causing BHK21cell to produce cytopathic effect. The cloned virus was identified by RT-PCR, indirect fluorescent immunoassay, plaque assay and other detection methods, and the results showed the infectious cDNA clone was obtained. One-step growth curve analysis shows the TCID50of the cloned virus is significantly lower than that of the parental virus, and there is no difference between the plaque morphology of these two.The successful construction of this full-length cDNA of the type Asia1FMDV strain within Jiangsu05lineages greatly simplifies the process of obtaining FMDV full-length cDNA clone. The infection cDNA can be used as a framework of genetic manipulation, which is of great help to the research of genetucally engineered vaccines with enhanced the virulence, strengthened stability and improved immunogenicity. Meanwhile, it is possible that we will be able to carry out the modification of the viral genome at the DNA level to research the structure and function related with RNA virus.