Construction And Optimization Of Vaccination Program Of Multi-Targets Attenuated Vaccines Based On Cucumber Mosaic Virus

After a plant is infected by a virus,it will trigger its own immune response,and the phenomenon that it can be protected from other strains of the same virus is called cross-protection.The use of weak virus（attenuated vaccine）is an effective measure for the prevention and control of plant virus diseases.However,there is currently a lack of multi-target attenuated vaccine that can effectively prevent tobacco virus diseases,and there is also a lack of a vaccination system with high vaccination efficiency when inoculating attenuated vaccines in the field.Cucumber mosaic virus has a wide range of hosts and serious damage.So,it is an excellent choice for the basic virus vector of attenuated vaccine.This study is based on the cucumber mosaic virus to create different types of bivalent and trivalent attenuated vaccines,and optimize the vaccination plan and vaccine vector.The vaccination plan was optimized through the analysis of three aspects that spray inoculation method,vaccine shelf life and the combined use of vaccine and fertilizer based on the existing attenuated vaccines in the laboratory.The success rate of Agrobacterium spray inoculation was 12.5%.The success rate of inoculating once was 80%and the success rate of inoculating twice could reach 100%after the tobacco leaf cutting treatment.One hour after leaf cutting,the success rate of inoculating once was 100%,it shows that cutting leaves is beneficial to vaccine vaccination.The carborundum was added into the Agrobacterium liquid to inoculate the tobacco without cutting leaves,and the success rate of inoculation was 93.3%,indicating that the addition of carborundum significantly improved the inoculation efficiency.There was little difference in the success rate of spray inoculation on the front and back of tobacco leaves.Therefore,the effective inoculation method is that spray inoculation on the front of the tobacco leaf in the state of leaf cutting once after Agrobacterium mixed with carborundum.Agrobacterium suspension liquid that stored at 28℃for 3 days can be successfully inoculated with tobacco.The mixed experiment of vaccine and fertilizer show that the use of foliar fertilizers reduces the effectiveness of vaccines,while the use of bio-organic fertilizer and calcium magnesium potassium phosphate have an additive effect on the effectiveness of vaccines.The recommended protocol for field vaccination is as follows:After the attenuated vaccine is mixed with carborundum,It has high efficiency to spray inoculate tobacco that are in the leaf-cutting state once or twice.Agrobacterium liquid is valid for 3 days.After vaccination,bio-organic fertilizer or calcium magnesium potassium phosphate can be applied to enhance the cross-protection effect of attenuated vaccine.On the basis of the early termination mutant pCC_FR2-2b PTII of 2b protein of CMV,the100 bp and 150 bp conserved fragments of different regions of potato X virus（PVX）were inserted into the mutants and obtained four PVX 100 insertion mutants and four PVX 150insertion mutants.These mutants were mixed with wild-type CMV_FnyRNA1 and CMV_FnyRNA3 to inoculate tobacco.The results showed that the insert sequences of all PVX 100 bp and 150 bp conservative fragment insertion mutants existed stably,and the tobacco showed weak pathogenic symptoms.It have a good cross protection effect on CMV and PVX.The cross protection effect of the mixed treatment of two,three,and four vaccines of the same type is gradually increasing.Use pCC_FR2-2b PTII as the base vector to insert the sequences of the three virus combinations,such as potato virus X（PVX）/potato Y virus（PVY）,potato virus X（PVX）/tobacco vein band mosaic virus（TVBMV）,potato virus Y（PVY）/tobacco vein band mosaic virus（TVBMV）.Each of the two viruses inserted 100bp of conserved sequences,and obtained 3 trivalent attenuated mutants(pCC_FR2-2b PTII-PX_5890-5989PY_617-716、pCC_FR2-2b PTII-PX_5890-5989TVB_7600-7699和pCC_FR2-2b PTII-PY_617-716TVB_7600-7699).These three target attenuated mutants were inoculated with wild-type CMV_FnyRNA1 and CMV_FnyRNA3,respectively,and tested for their efficacy against the target virus.The results show that the trivalent attenuated mutant pCC_FR2-2b PTII-PX_5890-5989PY_617-716is also resistant to CMV and PVX,but is not effective against PVY;pCC_FR2-2b PTII-PX_5890-5989TVB_7600-7699is also resistant to CMV and PVX,but is not effective against TVBMV;pCC_FR2-2b PTII-PY_617-716TVB_7600-7699can prevent CMV,but is not effective against PVY and TVBMV.The low fidelity of RdRp of RNA virus will cause the recovery of mutations,which is not good for the construction of attenuated vaccines.In response to this problem,CMV_FnyRdRp was modified to obtain high-fidelity RdRp and analyze the repair effect on mutations.Lysine 636 of the RdRp（2a）coding frame of CMV_FnyRNA2 was mutated into alanine,aspartic acid,arginine and tryptophan respectively and we obtained 4 mutants(pCC_FR2-2a-A₆₃₆、pCC_FR2-2a-D₆₃₆、pCC_FR2-2a-R₆₃₆、pCC_FR2-2a-W₆₃₆).The RdRp mutants was inoculated with wild-type CMV_FnyRNA1 and mutant CMV_FnyRNA3 respectively,and the results showed that:all RdRp mutants could not change the mutation recovery rate of mutant pCC_FR3-ΔCP_180F（100%）;RdRp mutants pCC_FR2-2a-A₆₃₆and pCC_FR2-2a-D₆₃₆cannot change the mutation recovery rate of mutant pCC_FR3-TLS_m1（100%）;RdRp mutants pCC_FR2-2a-R₆₃₆,pCC_FR2-2a-W₆₃₆can reduce the recovery rate of mutant RNA3-TLS_m1（from87.5%to 75%,50%）.These results indicated that the mutation repair rate of some mutants could be reduced to a certain extent by modifying RdRp.This study provides research materials and data support for the construction and application of attenuated vaccines based on CMV,providing methodological basis for field attenuated vaccine inoculation,and provides new research ideas and technical support for the prevention and control of plant virus diseases.