Nucleotide Sequences Analysis Of VP1 Gene Of Foot-and-Mouth Disease In Southern China Regions And Expression Of FMDV Capsid Precursor P1 In Feline Calicivirus

Foot-and-mouth disease is an acute and fever-causing zoonotic disease that infects such cloven-hoofed animals as swine, cattle and sheep by exposure to foot-and-mouth disease virus. The disease has severely decreased the productivity of domestic animals and restricted trades in animals and animal products between countries, due to the fact that it spreads quickly among a wide range of animals and is difficult to control. At present, vaccination is the main control strategy in our country.In this study, alignment analysis was conducted on the nucleotide sequences of the capsid proteins VP1 gene acquired from the foot-and-mouth disease virus O-ST-2010 strain in Guangdong 2010 and on the nucleotide sequences of VP1 gene of the epidemic strains gained in Southern China in previous years and the reference strains, and phylogenetic tree was built. The results indicate that similarity between O-ST-2010 strain and the strain O/HLJOC12/03 was the highest, reaching 93.8% and that of south-east Asia strains was between 92.2% and 83.3%, while the similarity of other genotype strains was no more than 85%. However, O-ST-2010 strain does not belong to the same genotype, according to topology, as strains prevalent in Southern China. The genotype of strains in Southern China in the past was part of Cathay or PanAsia Strain (classical China type or Middle East-South Asia type), but O-ST-2010 belongs to South-east Asia type. At present, FMD vaccine strains utilized in practice all belong to Cathay or Middle East-South Asia genotype. There is no vaccine that directs at South-east Asia genotype strains. Change of the genotype of prevalent strains will result in the change of virus antigenicity, which will lead to the decrease of protection provided by virginal vaccines, posing a severe challenge to FMD control in South China. It is urgent to develop vaccine which aims at South-east Asia genotype strains.In order to estimate the feasibility of feline calicivirus as a genetic vector and open up a new way to develop recombinant live vector vaccines of FMD, P1 gene of FMDV was inserted into infectious clone vector of FCV to construct recombinant FCV. FCV is single stranded RNA virus containing a 7.8kb genome, belonging to Vesivirus family Calicivirade, a major and highly prevalent pathogen in cats. In order to verify whether there are eight amino acids in FCV 3C-like protease, cutting sites in FMDV P1 were mutated into FCV 3C-like protease recognition motif, namely, whether there are four aminoacid units each at the terminal N and terminal C. The mutated P1, namely P1M2 was respectively inserted into the middle and the end of the leader protein (LC) of FCV infectious cloning vector to construct chimeric vectors rmFCV-LC-P1M2 and rmFCV-EA-P1M2, then F81 cells were transfected with the recombinant vectors. The results were evaluated by indirect immunofluorescence assay (IFA), RT-PCR, SDS-PAGE and Western-blot. Also, the growth kinetics of two recombinant viruses and parental virus were compared. The result shows that cytopathic effect and fluorescence were seen in cells transfected with two recombinant plasmids. A 2232bp band was amplified from cell culture by RT-PCR. Two bands were showed in SDS-PAGE, one of which was P1 protein about weight 83kD and the other of which was capsid protein of FCV about weight 63kD. It suggests that independent PI protein was expressed from FCV genome based on 3C-like protease recognition cutting sites of P1 mutated by hands. Meanwhile, target protein bands were seen in Western-blot analysis. The growth kinetics curves of rmFCV-EA-P1M2 virus and parental virus were similar except rmFCV-LC-P1M2. All results demonstrate that P1 had been expressed from FCV genome successfully and that FCV could be utilized as a genetic vector to express exogenous genes. A new approach to develop live recombinant vetor vaccines for FMD has been established.