Rescue Of The Genetically Engineered Foot-and-mouth Disease Virus And Its Research Of Sublingual Immunization

Firstly, the purpose of this study is to clarify the structural features of foot-and-mouth diseasevirus (FMDV) genomes, relationships of the sequence variations with its structure-function, andmolecular phylogeny among FMDV. The identity analysis and multiple alignment of184FMDVgenome sequences were undertaken, respectively, by using the DNAstar and Clustalx packages.FMDV genome sequences showed the entire ORFs size range from6963to7120nt, which couldencode the polyprotein with2320to2339aa. The homology of these nucleotide and amino acidsequences were greater than or equal to77.6%and greater than or equal to78.3%among sevendistinct serotypes, respectively. The data reveal novel highly conserved genomic regions,indicating variability as well as novel viral genomic motifs with likely biological relevance.Insights into viral RNA sequence conservation and variability and genetic diversity in nature willlikely impact our understanding of FMDV infections, host range, and transmission.Secondly, The full-length cDNA of foot-and-mouth disease virus (FMDV) strain was construct,and thus to set up basis for investigating the infectivity of it s in vit ro RNA transcript, elucidatingthe mechanism of pathogenesis of FMDV infection, and developing novel vaccine against FMDV.Using the over-lapping primers, which cover the whole genome of FMDV strain, were designedaccording to the FMDV nucleotide sequence. After ext racting the total RNA of virus f rom theinfected suckling mice, we amplified five cDNA f ragment s of FMDV strain by reversetranscription PCR. These PCR product s and clone vector were digested with the uniquerestriction enzymes respectively, ligated into the genome cDNA in vitro, respectively. The poly(C)tract of FMDV was synthesized in vitro and then inserted into clone vector. Finally, the full-lengthcDNA strain was constructed, and was identified by PCR, restriction enzymes digestion, and thewhole genome sequence was sequenced. The covering the FMDV whole genome were amplifiedrespectively, and ligated into the FMDV full-length cDNA molecule, which was proved by itsnucleotide sequences. It was concluded from the results that the full-length cDNA molecule ofFMDV strain has been const ructed successfully. The results obtained from this research haveestablished the basis for illustrating the mechanisms of pathogenicity and virulence of FMDV.Thirdly, an experiment was carried out to investigate SL co-administration of the FMDVvaccine, the molecular adjuvant GNP can enhance mucosal immune responses in murine model.Then the mice were randomly divided into groups and they were SL immunized with FMDValone or co-immunized with the GNP molecular adjuvant on days0,14and28. Afterimmunization, mucosal sIgA,T cell proliferation and the expession of cytokines were deteced byELISA. Compared to the group immunized with FMDV alone, the group immunized with themolecular adjuvant was induced higher level of mucosal sIgA, and delivery of GNP significantlyenhanced the higher level of antigen specific T cell proliferation and higher expession IL-4. Theresults demonstrated that SL delivery of GNP as a mucosal adjuvant can enhance the antigen specific mucosal immuneFinally, we investigate the influence of chitosan on the immunoreaction of FMDV RNA vaccine.Balb/c mice were immunized with FMDV RNA vaccine with chitosan as deliver carrier by asingle intramuscular injection and oral. Naked RNA groups of different immunizing doses byintramscular injection, inactivated vaccine group and physiological saline group were applied ascontrol groups. Cellular immunity, humoral immunity and mucosal immunity were detected,respectively.Compared with FMDV RNA vaccine with chitosan as deliver carrier with naked RNAgroup by intramuscular injection, FMDV RNA vaccine with chitosan as deliver carrier byintramuscular injection induced stronger cellular and humoral immunity. This vaccine inducedsimilar cellular immune response compared with inactivated vaccine that induced strongerhumoral immune responses. Mucosal immunity was induced only by RNA vaccine with chitosanas deliver carrier. Chitosan had certain adjuvant’s ability. It has provided a new strategy to obstainmore effective FMDV RNA vaccine. Many factors affecting the immunity efficiency of vaccineneed to be optimiazed further for application of this vaccine generally.