Isolation And Identification Of Novel Variant IBDV And Expression Of VP2 Protein And Its Immunogenicity Analysis

Infectious bursal disease(IBD)is an acute and highly infectious disease caused by infectious bursal disease virus(IBDV).IBD mainly attacks immature B lymphocytes in the bursa of birds,causing tissue damage and immunosuppression in infected chickens,even immune failure,and increasing susceptibility to second pathogens and other infectious diseases.Very virulent IBDV(vvIBDV)was found in the world in the late 1980s,causing huge economic losses to the poultry industry with its high mortality rate and severe immunosuppression.With the use of vaccines and continuous improvements in poultry management,wIBDV is gradually being brought under control.However an atypical IBD in recent years has emerged on farms in China and the pathogen was first identified and named as novel variant IBDV(nVarIBDV).As the existing commercial vaccine did not match the antigenicity of nVarIBDV,it cannot be effectively prevented and controlled,leading to a certain range of nVarIBDV epidemics in China,which will become a new threat to the healthy development of poultry farming.1.Isolation,identification and pathogenicity of novel variant IBDVIn this study,the clinical samples were detected by RT-PCR,which suspected IBD symptoms collected from different regions.The sequences of the VP2 gene and the VP1 gene of the 11 samples showed positive for IBDV.Among them,nine were nVarIBDV,and the other two were vIBDV and aIBDV.A novel variant IBDV strain of IBDV-SN1905-2 was isolated by chicken embryos,which caused severe atrophy of the bursa of SPF chickens,and BBIX was<0.7.The histopathology showed necrosis of lymphocytes,loss of follicular atrophy and widening of the interstitium.The viral virulence of the IBDV-SN1905-2 was determined to be 10-5.7 for BID50 in the animal infection test.2.Whole genome cloning and analysis of a novel variant IBDV strain SN1905-2In order to further understand the molecular characteristics and variation of nVarIBDV,so as to achieve better prevention and control effect,the whole genome sequence of SN19052 was amplified and sequenced.The nucleotide sequence of IBDV-SN1905-2 was analyzed for genetic evolution and bioinformatics.The results showed that the A segment of IBDVSN1905-2 was 3260bp in length and had two complete reading frames encoding VP5 protein and VP2-VP4-VP3 protein,respectively.Segment B was 2827bp in length,with a complete reading frame,encoding VP1 protein.The results of nucleotide sequence homology analysis of A and B segments,phylogenetic tree analysis and key amino acid loci analysis suggested that the novel variant IBDV-SN1905-2 had 98.4%homology with other novel variant IBDV such as SHG19,compared with 81.2%-95.9%with other types of reference strains;IBDVSN1905-2 has not only the four characteristic amino acids 222T,249K,286I and 318D of the varIBDV,but also has the three characteristic amino acids 221K,256I and 299S of the nVarIBDV.Further analysis revealed that the IBDV-SN1905-2 isolate had a specific mutation in one amino acid in each of the A and B segments,the specific biological significance of which needs to be further investigated.3.Expression and immunity of VP2 protein of Novel variant IBDVThe recombinant protein GST-VP2 was successfully expressed in the E.coli system by using the recombinant plasmid pGEX-6P-1-VP2.The recombinant protein was purified using a GSTrap FF agarose gel column and the antigenicity of the GST-VP2 protein was tested using an agar gel diffusion assay,which showed that the GST-VP2 protein reacted specifically with IBDV standard positive sera,showing a clear precipitation line.GST-VP2 protein was mixed with adjuvant to immunize SPF chickens and challenged with IBDV-SN1905-2 strain to evaluate the immunoprotective effect of GST-VP2 protein against the novel variant of IBDV.The results showed that the GST-VP2 protein provided 100%protection against the novel variant strain of IBDV.The BBIX was<0.7 in both the negative control and GST-VP2 immunized groups,compared with BBIX<0.7 in the challenged control group and commercial inactived vaccine groups.Our results provided reference for the prevention and control of novel variant IBDV in the future.