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In our previous study, the differentially expressed proteins have been identified by proteomic analysis in total haemocytes of shrimp ( Fenneropenaeus chinensis ) after white spot syndrome virus (WSSV) infection. To further investigate the differential response of haemocyte subpopulations to WSSV infection, granulocytes and hyalinocytes were separated from healthy and WSSV-infected shrimp by immunomagnetic bead (IMB) method, respectively. Then two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to analyze the differentially expressed proteins in haemocyte subpopulations between healthy and WSSV-infected shrimp. The results of flow cytometry (FCM) showed that about 98% of granulocytes and about 96% of hyalinocytes in purity were obtained. Quantitative intensity analysis revealed that 26 protein spots in granulocytes and 24 spots in hyalinocytes were significantly changed post WSSV infection. Among them, 24 proteins in granulocytes and 23 proteins in hyalinocytes were identified by MS analysis, which could be divided into eight categories according to Gene Ontology. The identification of prophenoloxidase (proPO), proPO 2 and peroxiredoxin in WSSV-infected granulocytes was consistent with the facts that the proPO-activating system and peroxiredoxin were mainly existed in granulocytes. The phagocytosis of hyalinocytes seemed to be enhanced during the infection, because several proteins that involved in phagocytosis, including clathrin heavy chain, ADP ribosylation factor 4 and Alpha2 macroglobulin were up-regulated in hyalinocytes upon WSSV infection. Our results also reflected the vital biological significance of calcium ion binding proteins in granulocytes and ATPase/GTPase in hyalinocytes during WSSV infection. The data in this study verified the roles of granulocytes and hyalinocytes involved in WSSV infection, and differentially expressed proteins identified in granulocytes and hyalinocytes had a close correlation with their function characteristics. [ABSTRACT FROM AUTHOR]

A number of white spot syndrome virus (WSSV)-binding proteins have been identified previously in the hemocytes of Fenneropenaeus chinensis. In order to further investigate the differential WSSV-binding proteins in hemocyte subpopulations, granular hemocytes and hyalinocytes were sorted from WSSV-infected shrimp by immunomagnetic bead (IMB) method. The results of ELISA and immuno-dot blot assay showed that the WSSV-binding activity of granular hemocytes proteins was much stronger than that of hyalinocytes proteins. And the percentage of WSSV-positive granular hemocytes was significantly higher than that of hyalinocytes post WSSV infection, indicating that granular hemocytes were more susceptible to WSSV infection. Moreover, a total of 9 WSSV-binding proteins were successfully identified in granular hemocytes and hyalinocytes by two-dimensional virus overlay protein binding assay (2D-VOPBA) and MALDI-TOF MS analysis, of which 3 binding proteins (arginine kinase, protease 1 and transglutaminase) existing in both hyalinocytes and granular hemocytes and 6 proteins (F 1 ATP synthase β-chain, hnRNPs, GAPDH, RACK1, β-actin and cellular retinoic acid) detected only in granular hemocytes. Among these identified WSSV-binding proteins, the transglutaminase (TG) was further recombinantly expressed, and the recombinant TG could be bound with WSSV. Subsequently, quantitative real-time PCR analysis showed that differential expression levels of WSSV-binding proteins were observed in granular hemocytes and hyalinocytes. The results of this study revealed that the WSSV-binding proteins were differentially expressed in granular hemocytes and hyalinocytes, which provided a deeper insight into the interaction between WSSV and hemocyte subpopulations. • Granulocytes proteins exhibited higher WSSV-binding activity than that of hyalinocytes. • A total of 9 WSSV-binding proteins were identified in granulocytes and hyalinocytes. • 3 WSSV-binding proteins were identified in both granulocytes and hyalinocytes. • 6 WSSV-binding proteins were detected in only granulocytes. [ABSTRACT FROM AUTHOR]