Construction And Immune Effect Evaluation Of Genotype ? ?? Bivalent Newcastle Disease Virus-like Particles

Newcastle disease(ND)is an acute highly contagious disease caused by Newcastle disease virus(NDV).The mortality rate in susceptible poultry is as high as100%,causing serious economic losses to poultry trade a listed disease of around the world.ND is one of the animal diseases that must be reported by the Office International Des Epizooties(OIE)and a major epidemic disease in China.In recent years,China's overall deployment prevention and control efforts have increased,and the epidemic situation has remained at a relatively low level.However,controlling and eradicating ND is not an easy task.The current prevention measures mainly rely on traditional vaccination,which cannot prevent the virus from replicating,resulting in serious virus contamination in local areas,and the epidemic is persistent and endemic.The commonly used NDV differential diagnosis method cannot distinguish antibodies produced by vaccine immunization from antibodies produced by infection,it is very unfavorable for epidemiological surveillance.Therefore,the development of a safe and efficient new vaccine,which can effectively identify wild-type infected antibodies and vaccine immune antibodies,is essential for the prevention and control of ND.Virus-like particles(VLPs)are nano-sized hollow particles assembled from one or more viral structural proteins.VLPs can be effectively recognized by natural immune cells,inducing excellent innate immune responses and adaptive immune responses.Moreover,VLPs do not contain viral genome,and have high safety,which become a hotspot in the field of vaccines in recent years.Based on a VLPs production platform,this study developed a chimeric VLPs based on the ND classic commercial vaccine(LaSota strain,genetype II)and a predominant genotype(NA-1 strain,genetype VII).Using the insect baculovirus expression system,production of bivalent NDV VLPs contains M protein of LaSota strain as skeleton,protective antigen protein F and HN of LaSota strain and NA-1strain as surface display.The immune efficacy of the VLPs were evaluated from four aspects:immune index,protection rate,viral load,virus shedding.As NDV VLPs only contain M,F and HN,another virus protein NP was used as a target to establish an ELISA method for differential diagnosis of ND wild-type infection and VLPs vaccine immunization.This project provides a safe and efficient candidate vaccine for the elimination and control of ND and provides a new strategy for the differential diagnosis of wild-type infectious antibodies and vaccine immune antibodies.1.Construction and identification of bivalent Newcastle disease virus-like particlesUsing the extracellular domain strategy,the extracellular domain of the F and HN genes of the NA-1 strain was ligated to the intracellular domain and the transmembrane domain of the LaSota strain F and HN by overlap extension PCR and designated as the rNA-1 F/HN gene.Recombinant baculovirus rBV-LaSota M,rBV-LaSota F,rBV-LaSota HN,rBV-rNA-1 F and rBV-rNA-1 HN were constructed using the Bac to Bac expression system.After co-infection of the five recombinant baculoviruses,the supernatant of sf9 cells was collected,purified by ultracentrifugation in combination with sucrose density gradient centrifugation.The product was identified by SDS-PAGE,Western Blot and transmission electron microscopy.The results showed that the target proteins were correctly expressed and assembled into VLPs,which is similar in size to the wild-type NDV,indicating that the bivalent NDV VLPs containing the LaSota strain M,F,HN protein and NA-1strain F and HN proteins were correctly constructed.2.Evaluation of bivalent VLPs immune effectSPF chickens were used immunized with different doses of bivalent NDV VLPs by intramuscular injection.Compared with traditional attenuated commercial vaccines,HI test results showed that the bivalent NDV VLPs can stimulate higher levels of HI antibody titer.Flow cytometry analysis showed that the bivalent NDV VLPs can effectively induce more CD3~+CD4~+T cells and CD3~+CD8~+T cells.In the face of virulent strains,the bivalent NDV VLPs can provide complete protection and has obvious advantages in detoxification time and viral load.3.Establishment of a differential diagnosis ELISA for distinguishing NDV VLPs vaccine antibodies from wild-type infectionsNDV NP protein was prepared by prokaryotic expression system,purified by affinity chromatography and identified by SDS-PAGE and Western Blot.The results showed that the NP protein was of high purity and could be used in subsequent experiments.Subsequently,an indirect ELISA method was established with NP protein as the target.The conditions such as antigen coating amount,sample dilution factor and antibody working concentration were optimized,and the performance of the detection method was evaluated.The results showed that the established ELISA method has high sensitivity,specificity and reproducibility,and can accurately distinguish serum samples of NDV VLPs vaccine immunization and wild-type infection subjects.In summary,this study successfully developed bivalent NDV VLPs containing both LaSota strain and NA-1 strain.The bivalent NDV VLPs can induce higher levels of humoral and cellular immune responses,providing full protection against homologous and heterologous virulent strains,and is superior to traditional commercial vaccines in terms of viral load and virus shedding period.In addition,the indirect ELISA method based on NDV NP protein can accurately differentiate the NDV VLPs immunization from NDV infection.All of these were laid a foundation for the prevention and eliminition of ND.