Isolation And Identification Of A Very Virulent Strain Of IBDV And Immunogenicity Analysis Of VP2 Protein Subunit Vaccine

Infectious Bursal Disease（IBD）is an acute and highly transmissible immunosuppressive disease caused by the infectious Bursal disease Virus（IBDV）,which is mainly characterized by the attack on the bursae,the central immune organ of chicks.Since its discovery,IBD has been widely prevalenting around the world.With the emergence of IBDV variants,traditional vaccines cannot produce effective immune protection,resulting in serious economic losses to the poultry industry.In this study,the bursal samples from chickens with suspected IBD in a chicken farm in Suihua of Heilongjiang Province were collected.Based on the overlapping regions of IBDV VP2 and VP5gene sequences in Gen Bank,a pair of identification primers were designed for PCR amplification.A specific band about 300 bp was amplified from one sample,which was consistent with the expected size and was correctly identified by sequencing.It was preliminarily identified as IBDV.The IBDV positive bursal sample was inoculated into chicken embryos for virus isolation,and the thickened chorioallantoic membrane,edema,bleeding of the embryonic body and other typical IBD lesions appeared on chicken embryos.The isolated virus was purified on LMH cells by plaque purification.The purified virus was further tested by AGP,serologic specificity determination,hemagglutination test,test for virus contamination,PCR identification,analysis of genetic evolution and amino acid homology analysis of VP2 gene.AGP showed that the purified virus was able to bind to the specific serum against IBDV to form specific precipitate lines.Neutralization experiments showed that the purified virus was completely neutralized by IBDV-specific serum.Hemagglutination test and test for contaminated virus showed that the purified virus was not hem-agglutinable and had no exogenous virus contamination.The purified virus was identified by PCR and sequencing,named as the IBDV-SH strain.The VP2 gene of the isolated strain was amplified and analyzed.The results showed that the isolated strain was in the same branch with the strains OKYM and HK46 of vv IBDV.VP2 amino acid sequence homology analysis revealed that the VP2amino acid sequence had high homology with vv IBDV strains,among which the homology reached to 100%with OKYM,HLJ-7,UK661,HK46 and Gx.This result implicates that the isolated strain IBDV-SH is a vv IBDV.To analyze the pathogenicity of IBDV-SH,10-day-old SPF chicken embryos and 6-week-old SPF chickens were infected with this virus.The results showed that the median embryo lethal dose(ELD₅₀)for chicken embryos was 10^-5.63/0.2m L,the median lethal dose(LD₅₀)for chickens was 10^-2.6/0.1m L and the median infective dose for chickens was 10^-4.33/0.1m L.The purified virus was inactivated and immunized 21-day-old SPF chickens,serum samples were collected 21 days after immunization,and the antibody AGP titer to be 1:12.8.At the same time,the immunized chickens were infected with IBDV-SH strain at 100 BID per chicken.The results showed that 80%of the immunized chicken were protected from disease,indicating that the IBDV strain had preferable immunogenicity.Furthermore,the VP2 gene of IBDV-SH was cloned into the prokaryotic expression vector p ET-28a and transformed into E.coli to induce the expression of VP2 protein.By AGP detection,the expressed recombinant protein reacted with IBDV antibody to form a specific precipitation line.Western blot analysis exhibited that a specific bands appeared at the position of 50 k Da,which was consistent with the size of the expressed protein and were indicated that the recombinant VP2 mainly expressed in soluble form.The concentration of inducer lactose,the induction time and,temperature,and the induction initiation bacteria concentration were optimized.It was determined that the concentration of the inducer lactose was 0.03 mol/L,the induction time was 5 h,the bacteria concentration at the initial inducing time was 0.5～0.6 of OD₆₀₀,the induced temperature was 37℃.By selected precipitation of the recombinant VP2 with different saturation levels of ammonium sulfate,the results showed that 40%saturated ammonium sulfate could precipitate all VP2 proteins after pre-treated with 20%saturated ammonium sulfate to removal some impure proteins,the expressed VP2 were shown largely purified when examined by AGP and SDS-PAGE.VP2 protein with AGP titers of 1:8,1:16 and 1:32 were taken to prepare a vaccine after inactivation and emulsification.The vaccine was examined for formulation,sterility,stability and safety,all of which met the required regulations.The prepared subunit vaccine and an IBDV commercial vaccine were respectively inoculated to 21-day-old SPF chickens.the AGP titer of serum antibody was measured 21 days after immunization.The AGP titer of the commercial vaccine group was 1:10.6,and VP2 protein vaccine groups were 1:6.1,1:8.0 and 1:18.4,respectively.No AGP titers was detected in the serum of the control group.Infection with an IBDV strain BC6/85by eyedrops,the results showed that the challenge protection rates of recombinant VP2 protein immunization group with AGP titer of 1:8,1:16 and 1:32 were 60%,80%and 100%,respectively.No pathological changes were found in tissues and organs of healthy chickens by autopsy.In the group of virus control,hemorrhage of pectoral muscle and leg muscle,jelly-like lesions,hemorrhage,atrophy or enlargement of bursal were observed.There were no obvious pathological changes observed in the immunized groups.Histopathological analysis revealed that the number of lymphocytes in the follicles of the bursal was reduced in the virus control group.Some epithelial cells of thymus medullary showed degeneration and necrosis.A large number of lymphocytes were necrotic in the red and white pulp of spleen.No significant histopathological changes were observed in immunized groups and the healthy control group.In conclusion,in this study,we isolated a very virulent strains of vv IBDV,expressed a recombinant VP2 protein of the virus in E.coli,and analyzed the immunogenicity of the expressed VP2 protein.These results placed a foundation for the development of recombinant subunit vaccines for effective prevention of IBDV and its variants.