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White spot syndrome virus (WSSV) is one of the most devastating viral pathogens of cultured shrimp worldwide. Recently published papers show the ability of WSSV structural protein VP28 to vaccinate shrimp and raise protection against the virus. This study attempted to identify the joining proteins of the aforementioned shrimp quasi-immune response by proteomic analysis. The other envelope protein, VP36B, was used as the non-protective subunit vaccine control. Shrimp were intramuscularly injected with rVPs or PBS on day 1 and day 4 and then on day 7 their gill tissues were sampled. The two-dimensional electrophoresis (2-DE) patterns of gill proteins between vaccinated and PBS groups were compared and 20 differentially expressed proteins identified by mass spectrometry, some of which were validated in gill and hemocyte tissues using real-time quantitative RT-PCR. Many of identified proteins and their expression levels also linked with the shrimp response during WSSV infection. The list of up-regulated protein spots found exclusively in rVP28-vaccinated shrimp include calreticulin and heat shock protein 70 with chaperone properties, ubiquitin, and others. The two serine proteases, chymotrypsin and trypsin, were significantly increased in shrimp of both vaccinated groups compared to PBS controls. The information presented here should be useful for gaining insight into invertebrate immunity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although adaptive immunity is believed to exist only in higher vertebrates, recent studies showed the ability to vaccinate shrimp and other crayfish against white spot syndrome virus (WSSV). This study attempted to establish parameters of vaccination coordinated with subsequent viral challenge to gain insights into the mechanisms of the protective response of penaeid shrimp. Two WSSV envelope proteins, VP28 and VP36B, were used as subunit vaccines expressed in Escherichia coli followed by histidine-tag affinity chromatographic purification. Shrimp vaccinated with the recombinant WSSV proteins and challenged with diluted WSSV inocula were intramuscularly injected in order to give a precise load. Results of the viral challenge trials showed complete survival in the rVP28 group in contrast to the rVP36B and PBS groups which exhibited 100% mortality. But this effective protection was exclusively induced from a combination of a higher dosage of rVP28 and a lower viral challenge pressure. The innate immune parameters analyzed among the three groups revealed that rVP28-treated shrimp showed the highest activity level (p<0.05) of phenoloxidase and superoxide dismutase during the entire period of 7 days post-vaccination. But there were no significant differences (p>0.05) in mRNA abundances of the Down syndrome cell adhesion molecule (Dscam) among all groups. In addition, total hemocyte counts significantly decreased in shrimp treated with the recombinant viral proteins compared to the PBS group. These results indicated that the existence of structure- and dose-dependent protective responses and the elevated innate immunity in shrimp following a protein-based vaccination might be responsible for conferring resistance against WSSV., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.

One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.