The protein kinase A-mediated phosphorylation of VpratSer79 site was found to be crucial for cell cycle arrest in HIV infection. All these examples illustrate that phosphorylation of viral proteins plays important roles in regulating processes such as gene expression, viral replication and cell cycle arrest during viral infection. Other than being reported as a serine-protease and an intracellular tubule type II, the VP4 protein of IBDV was also found to have novel roles as a biomarker for discriminating between pathogenic and nonpathogenic IBDV infections and an inhibitor suppressing the expression of type I interferon via interaction with the glucocorticoid induced leucine zipper. In our previous proteomic analysis of IBDV-infected cells, different protein spots of VP4 were evident in the two-dimensional electrophoresis gel. As it was of Ropivacaine hydrochloride interest to learn whether these spots represented post-translational modifications, in this study we identified the phosphorylation sites of VP4 and generated monoclonal antibodies against phospho-and nonSRPIN340 phospho-VP4 protein. Additionally, an in-depth analysis of the protease activity of phospho-VP4 was conducted. The phosphorylation of viral proteins has not been reported for all members of the family Birnaviridae. In the present study, the Western blot results showed that the VP4 is abundant in all cell fractions, including the nucleus, cytoplasm, membrane and in soluble cytoskeleton, and pSer538-VP4 accounted for as mall proportion of the insoluble cytoskeletal fraction. Correspondingly, in the 2-DE blots, the abundant VP4 protein and as mall amount of pSer538-VP4 protein were detected in the same protein spot, as well as in two different protein spots. Further immunofluorescence analysis revealed co-localization of theVP4 proteins recognized by the pSer538 and P530 mAbs. Similarly, previously published proteomic data have shown different 2-DE protein spots representing the viral VP4 protein in IBDV-infected CEF cells and bursallymphocytes. These results demonstrated that the VP4 in IBDV-infected cells with pSer538 modification was a minor and insoluble protein with different isoelectric points.