Construction Of Multi-Targets Attenuated Vaccine From Cucumber Mosaic Virus And Assays On The Optimization Of Vaccine Application Scheme

Plant virus disease is one of the most serious diseases affecting plant growth,reducing crop yield,affecting quality and causing huge losses to our agricultural economy.Attenuated vaccine cross-protection is an effective measure to prevent plant virus diseases,but vaccines have limitations such as single dosage form,short shelf life,and limited target viruses that can be controlled by a single dose of vaccine.Cucumber mosaic virus（CMV）is one of the most host-rich and widely distributed plant viruses,and its multi-segmental genome provides multiple site-specific options for mutagenesis,which has great potential for the development of attenuated vaccines.In this study,CMV was used as the basic vaccine vector backbone to construct double,triple,quadruple targets attenuated vaccines,while comparing the shelf life of vaccines with different cryopreservation methods to provide attenuated vaccine materials and feasible solutions to extend the shelf life for the control of plant virus diseases.The CMV_FnyRNA2 attenuated mutants p CC_FR2-2b PTII and p CC_FR2-2b PTIII constructed were used as vaccine basic vectors.Four kinds double targets attenuated mutants were constructed by inserting 100 bp virus fragments(TVBMV_7600-7699,TVBMV_7700-7799,TVBMV_8880-8979,TVBMV_8980-9079)of different conserved regions into the attenuated mutant p CC_FR2-2b PTII.Two kinds triple targets attenuated mutants were constructed by inserting221 bp fragments(TVBMV_7600-7699TMV_2158-2278,TMV_2158-2278TVBMV_7600-7699)from TVBMV and TMV into the attenuated mutant p CC_FR2-2b PTII.Six kinds quadruple targets attenuated mutants were constructed by inserting a total of 321 bp of viral linker fragments of TVBMV,TMV and PVY in different order(TMV_2158-2278PVY_517-616TVBMV_7600-7699,TMV2158-2278TVBMV7600-7699PVY517-616,PVY517-616TVBMV7600-7699TMV2158-2278,PVY517-616T-MV2158-2278TVBMV7600-7699,TVBMV7600-7699PVY517-616TMV2158-2278,TVBMV_7600-7699TMV_2158-2278PVY_517-616)on the attenuated mutant p CC_FR2-2b PTIII.Each mutant was mixed with CMV_Fnywild-type RNA1 and RNA3 respectively and inoculated into Nicotiana benthamiana,showing weak pathogenicity and good genetic stability.The single or mixed challenge inoculation of the target virus after 7 days of pre-inoculation of the above mutants showed the best control effect on CMV（72.0%～100%）,and also showed a certain control effect on each target virus（7.4%～46.2%）.In the cross-protective effect experiment of double targets attenuated mutants,the cross-protective effects on TVBMV of four insertion mutants of TVBMV were significantly different.The mutants inserted with TVBMV_7600-7699fragment had the best control effect on TVBMV and mixed virulent virus,which were 37.0%and 22.2%.In the cross-protection experiment of triple targets attenuated mutants,it showed good cross-protection effects on TVBMV,TMV and mixed virulent viruses,which were respectively 40.0%～44.4%,32.0%～37.0%and 24.0%～32.0%.There was no significant correlation between the control effect on target viruses and the insertion sequence of virus fragments.In the cross-protection experiment of quadruple targets attenuated mutants,the six mutants showed good cross-protection against CMV,TVBMV,TMV and PVY infection alone or in combination,and the control effects on each target virus infection alone were respectively 87.0%～100%,22.2%～46.2%,22.2%～44.4%and 28.5%～44.4%.The control effect on mixed virulent strains was 16.0%～32.0%.There was no significant correlation between the control effect on target viruses and the insertion sequence of virus fragments.In order to guide the field application and reduce the cost of vaccine use as much as possible,it is necessary to determine the initial concentration range of the vaccine.The dilution limits of the mutant p CC_FR2-2b PTII and the mutants inserted with 100 bp and 221 bp virus fragments(OD₆₀₀=1.2)were analyzed.The p CC_FR2-2b PTII mutant was diluted 10¹-10⁵times and inoculated.It still had good genetic stability,and the disease index was 3.7～26.0,maintaining the attenuated characteristics.The mutant inserted into the 100 bp heterologous virus fragment began to show a certain proportion of mutation recovery after dilution 10⁴times,and basically recovered completely after dilution 10⁵times.The disease index reached48.1,showing a certain strong virus characteristics.The mutant inserted with 221 bp heterologous virus fragment began to appear mutation recovery after dilution of 10³times,and with the increase of dilution times,the recovery was more complete.The disease index of inoculation reached 70.4 after dilution of 10⁵times,which was recovered to strong virus.In conclusion,the over-diluted initial inoculation concentration during vaccine use will lead to a decrease in vaccine stability.The initial concentration of OD₆₀₀=1.2 of the vaccine bacterial solution in the field during the use of the recommended dilution should not exceed 10²times.In order to reduce the cost of preservation and long-distance transportation of large-volume bacterial liquid vaccines,this study compared and analyzed the vaccine stability and cross-protection effects of three cryopreservation methods（Centrifugal preservation method,Direct freeze-drying preservation and Freeze-drying preservation after centrifugation）of bacterial liquid attenuated vaccines.Centrifugal preservation method:The vaccine bacteria without supernatant after high-speed centrifugation were preserved at two groups of low temperatures（4℃,-20℃）for 15 days,30 days and 60 days before re-suspension inoculation.All showed good genetic stability and good cross-protection against CMV.With the prolongation of preservation time,the relative control effect gradually decreased.The relative control effects were 74.1%,51.9%and 51.9%after 15 days,30 days and 60 days at 4℃,74.1%,59.3%and 44.4%after 15 days,30 days and 60 days at-20℃.When stored at 28℃for 15 days and 30 days,the vaccine still had good stability,and the cross-protection effect on CMV was worse than that of the two groups,only 37.0%and 22.2%.After 60 days of storage,it had completely returned to the wild type,and basically no longer showed obvious cross-protection effect,with a relative control effect of only 7.4%.Direct freeze-drying preservation method:The vaccine bacterial powder treated by freeze-drying machine was stored at 4℃,-20℃for 30 days and 60 days,and then re-suspended and inoculated.All of them had good genetic stability,and showed very good cross-protection against CMV.The relative control effect was above 88.0%.Centrifugation and freeze-drying preservation method:The vaccine bacterial powder treated by freeze-drying machine after high-speed centrifugation to remove the supernatant of the bacteria was stored at 4℃,-20℃for 30 days and 60 days,and then re-suspended with sterile water,pure water and tap water,respectively.All have good genetic stability,and the relative control effect of each treatment group on CMV reached 72.0%after 30 days of storage.After 60 days of storage,resuspension with sterilized water was better than pure water and tap water.The relative control effect at 4℃（66.7%～100%）was higher than that at-20℃（66.7%～74.1%）.The cryopreservation method of the above three bacterial liquid vaccines solves the problem that the bacterial liquid vaccines are not easy to be stored in large volume,improves the convenience of long-distance transportation,and also reduces the cost.In this study,12 kinds of multi-targets attenuated vaccines with genetic stability and resistance to multiple viruses were screened,which provided vaccine materials for the prevention and treatment of corresponding plant viral diseases.At the same time,the stability analysis of vaccine after dilution and the comparison of three cryopreservation methods provide data support for reducing the cost of use,preservation and transportation during vaccine application.