Analysis and identification of tyrosine phosphorylated proteins in hemocytes of Litopenaeus vannamei infected with WSSV.

Previous studies have demonstrated that protein tyrosine phosphorylation plays an important role in WSSV infection. In the present work, in order to further elucidate the potential role of protein tyrosine phosphorylation in white spot syndrome virus (WSSV) infection. The expression variation of tyrosine phosphorylated proteins in hemocytes of shrimp (Litopenaeus vannamei) after WSSV infection were examined by flow cytometric immunofluorescence assay (FCIFA) and enzyme linked immunosorbent assay (ELISA), and results showed that the level of protein tyrosine phosphorylation in hemocytes fluctuated significantly after WSSV infection and exhibited two peaks at 6 and 24 h post infection (hpi). Meanwhile, tyrosine phosphorylated proteins in hemocytes after WSSV infection were also detected by cell immunofluorescence, and results showed that the fluorescence intensity in hemocytes was altered with the course of WSSV infection and showed stronger fluorescent signals at 6 and 24 hpi compared to other time points. Furthermore, two dimensional gel electrophoresis (2-DE) and 2-DE western blotting were applied to identify the differentially expressed tyrosine phosphorylated proteins in hemocytes before and after WSSV infection. The result of 2-DE western blotting showed that there were nine tyrosine phosphorylated proteins in the hemocytes of healthy shrimp, whereas twenty-one tyrosine phosphorylated proteins were detected in the hemocytes of shrimp at 6hpi. Then, the differential tyrosine phosphorylated proteins were analyzed by Mass Spectrometry (MS), and eight of them were identified to be sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, actin-D, phosphopyruvate hydratase, beta-actin, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase. Moreover, the expression levels of sodium/potassium-transporting ATPase subunit alpha, ubiquitin/ribosomal L40 fusion protein, phosphopyruvate hydratase, ATP synthase subunit beta, receptor for activated protein kinase c1 and protein disulfide-isomerase were examined to be up-regulated post WSSV infection by quantitative real-time RT-PCR. Taken together, these results demonstrated that protein tyrosine phosphorylation was involved in the process of WSSV infection, which might play an important role in the immune response to WSSV infection in shrimp.

The Roles of ß-Integrin of Chinese Shrimp (Fenneropenaeus chinensis) in WSSV Infection.

Our previous study demonstrated that an integrin ß subunit of Chinese shrimp (Fenneropenaeus chinensis) (FcßInt) plays an important role in white spot syndrome virus (WSSV) infection. In the present work, in order to further elucidate the potential role of FcßInt in WSSV infection, the recombinant extracellular domain of ß integringene of F. Chinensis (rFcßInt-ER) was expressed in Escherichia coli BL21 (DE3), and the eukaryotic expression plasmid PcDNA3.1-FcßInt-ER (PFcßInt-ER) was also constructed. Far-western blotting was performed to determine the binding specificity of rFcßInt-ER to WSSV envelope proteins, and results showed that rFcßInt-ER was able to specifically interact with rVP31, rVP37, rVP110 and rVP187. Moreover, the blocking effects of mouse anti-rFcßint-ER antibodies were both detected in vivo and in vitro. The ELISA and Dot-blotting in vitro assays both showed that mouse anti-rFcßInt-ER antibodies could partially block the binding of WSSV to the hemocyte membrane of F. chinensis. In the in vivo assays, the mortality of shrimp injected with WSSV mixed with anti-rFcßInt-ER antibodies was delayed, and was lower than in the control group. While the shrimp were intramuscularly injected with PFcßInt-ER, transcripts of PFcßInt-ER could be detected in different shrimp tissues within 7 days, and the mortality of shrimp injected with PFcßInt-ER was also delayed and lower compared with the control group post WSSV challenge. Furthermore, gene silencing technology was also used to verify the effect of FcßInt in WSSV infection, and results showed that the expression levels of the WSSV immediate early gene iel, early gene wsv477, and late gene VP28 and the mortality of F. Chinensis were all significantly decreased in the FcßInt knock-down hemocyctes compared to the control group. Taken together, these results suggest that FcßInt plays important roles in WSSV infection.