Construction And Characteristics Analysis Of Genetically Engineered Type O Foot-and-Mouth Disease Virus With Increased Thermostability

Foot-and-mouth disease(FMD),of which foot-and-mouth disease virus(FMDV)is the causative agent,is a highly contagious and devastating disease of cloven-hoofed animals including cattle,pigs,sheep.It is mainly animal disease for prevention and control of the national long-term plan for the prevention and control of animal epidemics.Regular vaccination with inactivated vaccines is the principal means for prevention,control and eradication FMD.However,FMDV is extremely sensitive to thermal dissociation(especially type O FMDV).The virions easily dissociate into pentamer subunits(12S)when subjected to moderate heating,leading to obviously reductions of effective antigen content(146s)during storage and transportation.Consequently,vaccines derived from heat labile FMDV severely limit protective efficacy of vaccine and will further effect prevention and control of FMD.Therefore,there is an urgent need for developing of DIVA(differentiating infected from vaccinated animals)vaccine with increased thermostability to effectively improve the quality of FMD vaccine and meet the technical requirements for the national prevention and control of FMD.In order to develop FMD DIVA vaccine candidates with improved thermostability,in this thesis,three parts of works have been investigated.1.Using a previously constructed type O FMDV full-length infectious clone pO/TAA,10 recombinant full-length plasmids containing a single or multiple amino acids substitutions in the structural protein were constructed.The Not I-linearized recombinant plasmids were transfected into BSR/T7 cells expressing T7 RNA polymerase to rescue the recombinant viruses.9 genetically engineered FMDVs were successfully recovered.The recombinant viruses were analyzed by RT-PCR,sequencing and indirect immunofluorescence and the results showed that the six viruses(rvO/EN,rvO/ER,rvO/FH,rvO/FI,rvO/FN,rvO/FO)contained expected amino acid mutations.2.Thermal and acid stabilities of the recombinant viruses under different temperature and pH were determined by thermal inactivation assay and the results demonstrated that the heat and acid stability of the recombinant viruses(rvO/EN,rvO/FN,rvO/FO)were significantly improved compared with the parent virus.The recombinant viruses were analyzed by one-step growth curves and plaque morphologies and the assays indicated that the single or multiple amino acids substitutions in the structural proteins affected the virus replication in different degrees.The substitution of the recombinant virus(rvO/EN)was partially recovered and the other mutations remained stable after 8 serial passages in BHK-21 cells.3.The inactivated vaccines prepared from three recombinant viruses with increasing thermostability were immunized pigs,respectively.The serum samples were assayed by virus neutralization assay at 0,7,14,21,28,35,42 and 49 days post-vaccinated to analyze the effect of mutations in structural proteins for viral antigenicity.The results showed that the substitutions of S2093 H,Y2098F and S2093H-Y2098 F did not affect the immunogenicity of the recombinant FMDVs.