| Infectious Bursal Disease and Newcastle Disease are important viral diseases of poultry and other birds,threatening the development of the poultry industry and having important implications for poultry health security and the economy around the world.Infectious bursal diseases(IBD)are caused by the Infectious bursal diseases virus(IBDV).Chickens are prone to severe immunosuppression after infection with IBDV,making them highly susceptible to secondary infection,and unresponsive to vaccination with other pathogens.Infectious bursal disease vaccines are mostly developed based on classic strains,and cannot provide complete protection to chickens infected with clinically circulating super-virulent strains or mutant strains.Newcastle disease(ND)is an acute and highly contagious avian viral disease caused by a virulent strain of Newcastle disease virus(NDV).The main epidemic strain of Newcastle disease in my country is genotype VII,and the main vaccine strain is genotype II LaSota attenuated strain.The mismatch of genotypes and the unsafety of live vaccines make the purification of Newcastle disease difficult.Marketed vaccines can no longer meet the needs of various types and multi-genotypes of epidemic prevention and control.Green ecological,safe,and efficient new vaccines have become the development needs of the poultry farming industry and the key research direction of the national development strategy.Therefore,a safe and efficient new type of vaccine has been developed.Vaccines are necessary for the joint prevention and control of IBD and ND."Virus-like particles"(VLPs)do not have complete viral genomes and cannot replicate.They are non-infectious,more structured recombinant vaccines that can display densely repeated epitope characteristics in a limited space.,has the reputation of "strong activator" in stimulating the immune system,and can effectively stimulate humoral immunity,cellular immunity,and mucosal immunity.In addition,VLPs demonstrate extremely high advantages in terms of biosafety,immunogenicity,and carrier platform richness.To scientifically prevent and control IBDV and NDV,this study developed a green,safe and efficient infectious bursal disease-Newcastle disease dual chimeric virus-like particles(IBD-ND cVLPs)based on the Newcastle disease virus-like particle vector platform.)vaccine candidate strains.The chimeric virus-like particle envelope is based on the M protein of the genotype VII NA-1 strain as the backbone,and the outside contains the important protective antigens HN protein and F protein of the NA-1 strain and the candidate epitope VP2 protein of the IBDV epidemic strain SH99 strain.After purification,the intramuscular injection was used to immunize SPF chickens,and sampling was continued after the challenge to evaluate the immune effect of the vaccine from the aspects of cellular immunity,humoral immunity,challenge protection,in vivo viral load,and in vitro detoxification time.Vaccination of poultry with this safe and effective dual vaccine of IBD and ND has potential advantages,a single immunization protects chickens from both viruses,reduces vaccine costs and labor input,and provides protection against infectious bursal disease and Newcastle disease.VLP provides vaccine stockpiles for the control of infectious bursal disease and Newcastle disease.1.Construction and identification of IBD-ND cVLPsBased on the characteristics of the type II transmembrane protein of NDV HN protein and the replacement pattern of the extracellular domain,the overlap extension PCR technique was used to connect the intracellular domain and transmembrane domain of the HN protein of the NA-1 strain to the VP2 protein of the SH99 strain into a flexible peptide.Named rBV cHN+VP2.Sf9 cells in suspension culture were infected with rBV cHN+VP2 together with rBV-M,rBV-HN,and rBV-F constructed and preserved in the laboratory,and they were fully self-assembled.After centrifugation and purification,they were identified by Western Blot and transmission electron microscopy.The results showed that the components of cVLPs were expressed correctly and had capsule and fiber structures after assembly,indicating that the IBD-ND cVLPs containing the candidate epitopes of IBDV were correctly constructed.2.Evaluation of the immune effect of IBD-ND cVLPsCommercialized IBDV attenuated vaccine groups and commercialized NDV attenuated vaccine groups were immunized by eyedrops,and IBD-ND cVLPs were immunized by intramuscular injection.After immunization,experimental animals were challenged,and continuous sampling was performed to analyze body weight change,survival rate,viral load,detoxification time,cellular immunity,and humoral immunity activation status to evaluate the immune effect of IBD-ND cVLPs.The results show that the dual virus-like particle group can rapidly stimulate humoral immunity,and maintain the antibody level far above the level of immune protection for a long time.Group quite.Moreover,the lymphocyte proliferation experiment showed that there was no significant difference in the SI value between the dual viruslike particle group and the commercial vaccine group(p<0.5),both of which could effectively induce lymphocyte proliferation.In conclusion,this study correctly constructed IBD-ND cVLPs containing protective antigens of both the IBDV epidemic strain SH99 strain and the NDV domestic dominant epidemic strain NA-1 strain,and these cVLPs had good biological activity and biological morphology.Neutralizing antibodies can be induced after immunizing chickens.The detection of T lymphocyte proliferation ability shows that the cVLPs prepared in this experiment can produce good cellular immunity,and the time of in vitro detoxification and the viral load in vivo are reduced after challenge,which also shows that cVLPs can effectively Block virus replication.This study not only enriches the application of ND VLPs as a carrier platform but also provides a green,safe and efficient novel vaccine candidate strain that can simultaneously prevent IBD and ND. |
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