Isolation And Identification Of IBDV Epidemic Strain And Study On The Modulation Of IBDV VP3 Protein On Host IRF7 Pathway

Infectious bursal disease virus(IBDV)is the pathogen of Infectious bursal disease(IBD),which can exclusively target the B lymphocytes of the bursal and cause a contact and immunosuppressive infectious diseases in young chickens.In recent years,the epidemic strains of IBDV have shown new characteristics such as gene rearrangement,gene recombination and antigen variation,which have brought new challenges to the prevention and control of IBD.Therefore,continuous tracking of the molecular epidemic of IBDV strains and further exploration of the interaction between virus and host will lay a foundation for us to understand the epidemic trend of the strains,analyze the molecular pathogenic mechanism of the strains,and provide a reliable scientific basis for the research and development of effective IBD prevention and control measures.Firstly,the IBD suspected bursa of fabricius tissues collected from Guilin,Guangxi in 2019 were detected by RT-PCR based on IBDV-VP2 hypervariable region(v VP2).Four samples were selected for IBDV isolation and sequence analysis of v VP2 and VP1-b.The results showed that the nucleotide similarities of v VP2 genes between GL1902 and SHG352,SHG19 novel antigenic variants were up to 94.1%-96.6%,and the characteristic amino acid sites of v VP2 of GL1902were consistent with those of SHG19 and other novel variants.In the phylogenetic tree,GL1902 belongs to the same branch as SHG19,SHG382 and other novel variants.The nucleotide similarity of v VP2 gene between three isolates GL1906,GL1909,GL19010 and vv IBDV(such as UK661,HLJ-0504,NN1172)were up to95.0%-99.6%,and the characteristic amino acid(aa)at 222,256,284,294 and 299were the same as those of vv IBDV.However,The the aa at 279N of the three isolates were the same as the attenuated strain the phylogenetic analysis revealed that the three isolates,GL1906,GL1909,GL19010 belonged to the same branch as vv IBDV HLJ-0504 and NN1172.In the study based on VP1-b gene,the nucleotide similarities between GL1902,GL1909 and new antigen variant strain were up to95.7%-96.5%,and the characteristic aa sites of 242D,287T,390L,393E were consistent with new variants such as SHG19.In the phylogenetic tree,the two isolates GL1902,GL1909 belonged to the same branch as new variants such as SHG19 and SHG382.The nucleotide similarities of the other two isolates,GL1906and GL19010,with those of HLJ-0504,NN1172,and other vv IBDV isolates were88.1%-99.1%.The characteristic aa sites at 242D,287A,390L and 393E were the same as the reassortant strain such as NN1172 and HLJ-0504,and formed a unique branch with reference strains such as HLJ-0504 in the phylogenetic tree.These results indicated that GL1902 was a novel mutant with genotype A2d B1b.GL1906and GL1910 were the very virulent strains with genotype A3B3.GL1909 was a novel mutant reassortant with genotype as A3B1b.Secondly,the regulatory effect of vv IBDV infection on chicken interferon regulatory factor 7(IRF7)was studied.In the experiment,the relationship between IBDV and IRF7 signaling pathway were firstly analyzed in DF-1 cells.As a result,IBDV significantly increased IRF7 and IFN-?m RNA levels after 18 hour post-infection(hpi),and began to slightly down-regulate IRF7 and IFN-?m RNA levels after 18 hpi,while the viral replication level continued to increase and maintained until 24 hpi.Further,when higher viral infection dose was used,lower IRF7 and IFN-?m RNA levels were detected,suggesting that the inhibition of IRF7 m RNA expression was related to the viral infection dose.Western blotting analysis confirmed that the degradation of IRF7 was indeuced by IBDV at 18 hpi and afterwards,however,the protein degradation was not virus load dependent.Further studies showed that the dimerization level of IRF7 in IBDV-infected cells was significantly lower than that in the uninfected control group(P<0.05).It was also found that the expression level of IRF7 in IBDV-infected cells that was treated with MG132 was significantly higher than that in the untreated group(P<0.01),indicating that IBDV induced the degradation of IRF7 through the proteasome pathway.When IBDV infected the DF-1 cell with IRF7 overexpression,lower viral load was detected by QPCR as compared to the naked plasmid.Further,the IRF7protein was still degaraded in the overexpressed cells at 18 hpi after infected by IBDV,and reached the lowest protein level at 36 hpi.Finally,we transfected DF-1 cells with the eukaryotic expression plasmid of IBDV VP3 protein to investigate the effects of IBDV VP3 protein on the IRF7 and IFN-?signaling pathways in cells.The results showed that after the p3×Flag-CMV14-VP3 plasmid was transfected with 100 TCID₅₀ NDV stimulation,the m RNA and protein expression levels of IRF7 and IFN-?were significantly lower than those in the empty vector transfection group.At the same time,the dimerization level of IRF7 protein was affected by IBDV VP3.After the p3×Flag-CMV14-VP3 plasmid was transfected,the amount of dimerized IRF7protein in Poly(I:C)stimulated cells was significantly reduced.In Poly(I:C)stimulated cells,the expression of IRF7 protein in p3×Flag-CMV14-VP3transfected cells treated with the proteasome inhibitor MG132 was higher than that in the untreated cells,indicating that IBDV VP3 protein promotes the degradation of IRF7 through the proteasome pathway.In summary,four IBDV strains were isolated from Guilin,Guangxi Province in this study.Genetic sequence analysis showed that GL1902 was a novel mutant with genotype A2d B1b.GL1906 and GL1910 were the super virulent strains with genotype A3B3.GL1909 was a novel mutant with A3B1b genotype and a rearrangement type.The regulation of IRF7 by vv IBDV is firstly activated and then inhibited,thus affecting the expression of downstream IFN-?and thus the antiviral effect of cells.The VP3 protein of IBDV is involved in the regulation of IRF7.IBDV and its VP3 protein affect the transcription and expression level of IRF7 and IFN-?through the proteasome pathway.The higher the IBDV concentration is the smaller the m RNA expression levels of IRF7 and IFN-?.At the same time,the dimerization level of IRF7 protein is reduced due to the action of IBDV and its VP3 protein.When the IRF7 plasmid is transfected into IBDV-infected cells,the replication of IBDV is reduced.IBDV also causes the reduction of IRF7 protein expression in cells at the late stage of viral infection.The above experimental results have broadened the understanding of the epidemic characteristics and molecular pathogenic mechanism of IBDV,and provided a scientific basis for the prevention and treatment of IBD.