Identification And Vaccine Strain Construction Of The Novel Variant Infectious Bursal Disease Virus

Infectious bursal disease(IBD),caused by infectious bursal disease virus(IBDV),is an acute,highly contagious,and fatal infectious disease,which is the most important immunosuppressive disease threatening the poultry industry.Till now,very virulent IBDV(vvIBDV)infection is gradually being controlled in China.However,recently,a new situation emerged that some immunized chickens were continuously detected positive for IBDV.Although it did not kill chickens,it led to severe immunosuppression,reduced production performance of the flocks,resulting in severe economic losses.It is called atypical IBD,which has become a new threat to the healthy development of the poultry industry.However,its pathogen characteristics and epidemic situation are not clear.In view of this serious problem faced by the poultry industry,this study conducted an epidemiological investigation and took the lead in identifying the pathogen of atypical IBD as the novel variant IBDV,which was spreading in the main poultry farming areas in China.Furthermore,SHG19 strain was used as the representative strain to evaluate the pathogenicity of the novel variant IBDV in layers and broilers,respectively.The results showed that SHG19 strain could cause acute infection in chickens and damage the bursa(the central immune organ).At 4 days post-infection,the chickens were immunized with avian influenza bivalent inactivated vaccine(H5 + H7)and Newcastle disease vaccine(LaSota strain),respectively.The results showed that the HI antibody titer of the infected chickens was significantly lower than that of the control group.In addition,SHG19 strain infection also caused significantly decline in weight gain in broilers,and the weight of slaughter broilers was 16% lower than that of control broilers.In order to reveal the reason why the novel variant IBDV was circulating in immunized chicken flocks in China,the immunoprotective effects of three vaccines against vvIBDV on the the novel variant IBDV were evaluated in this study.The results showed that none of the three vaccines could produce completely effective protection against the novel variant IBDV.Serum cross-neutralization test found that the antigenicity between the novel variant IBDV and vvIBDV was significant different.The molecular basis of the antigenicity difference between the novel variant IBDV and vvIBDV was further determined in this study.The results showed that the novel variant IBDV had different monoclonal antibody(MAb)reactivity patterns from vvIBDV.The seven MAbs including 2-5C-6F only recognized vvIBDV but did not recognize the novel variant IBDV.The epitope identification results showed that the epitope recognized by neutralizing MAb 2-5C-6F was 317SGGQAGDQMSWSASGSLAVT336 of VP2.There are two amino acids(aa)differences of G318 D and D323 E in this epitope between vvIBDV and the novel variant IBDV.The further research found that double mutations of G318D/D323 E made vvIBDV Gx no longer be recognized by MAb 2-5C-6F,while the D318 G and(or)E323D mutation could make the originally unrecognized novel variant IBDV SHG19 be recognized by MAb 2-5C-6F.The double mutations of G318D/D323 E further made the originally unneutralized novel variant IBDV SHG19 be neutralized by MAb 2-5C-6F.These data prove that the aa 318 and 323 of VP2 are one of the key factors involved in the immune evasion of novel variant IBDV from vvIBDV antibodies.Aiming at the prevention and control of the new disease,a reverse genetic vaccine candidate strain rGtVarVP2 of the novel variant IBDV was constructed and rescued in this study,which used the genome of the attenuated vaccine strain Gt as the skeleton and the main protective antigen gene of the novel variant IBDV SHG19 replaced its corresponding fragment.The preliminary results showed that the reverse genetic vaccine candidate rGtVarVP2 of the novel variant IBDV could provide 100% immune protection against wild virus challenge.Targeting the urgent problems in the prevention and control of diseases in the poultry industry,we first identified that the pathogen of atypical IBD is the novel variant IBDV,and clarified its pathogenic characteristics such as tissue damage,immunosuppression,and immune interference,and revealed the main reason why the novel variant IBDV is spreading in immunized chicken flocks,and clarified the molecular basis of the antigenicity difference between the novel variant IBDV and vvIBDV,and preliminarily developed the candidate vaccine strains with good immune effect.This study is of great significance for IBD comprehensive control and healthy breeding.