SDS-PAGE and western blotting of the LCDV cellular receptor-27.8kDa protein.

<p>Lane 1: Molecular mass marker; Lane 2, 3: SDS-PAGE of FG and HINAE cell membrane protein, stained with coomassie bule; Lane 4, 5: SDS-PAGE of FG and HINAE cell cytoplasm protein, stained with coomassie bule; Lane 6, 7: reaction with anti-27.8R MAbs showed only one 27.8 kDa in FG and HINAE cell membrane protein; Lane 8, 9: reaction with anti-27.8R MAbs showed no band in FG and HINAE cell cytoplasm protein; Lane 10, 11: anti-WSSV MAb 1D5 instead of anti-27.8R MAbs served as negative controls.</p

Dynamic expression of 27.8R in FG (A) and HINAE (B) cells post LCDV infection detected by sandwich ELISA.

<p>The cells were infected with LCDV at a MOI of 3.0 and sampled at different time points post infection. Error bars represented SD. Data represented the absorbance value at 405 nm (mean ± SD; n = 3) and were compared by Student’s <i>t</i> test. Un-infected cells (0 h) represented the negative control. The asterisk represented the statistical significance (<i>p</i> < 0.05) as compared with the negative control.</p

Dynamics of LCDV copies in FG and HINAE cells post LCDV infection investigated by qPCR.

<p>(A) Standard curve of LCDV MCP qPCR assays. The X-axis showed the positive control plasmid copy number in Log 10 value, and the Y-axis indicated the corresponding cycle threshold (Ct) value. R²: coefficient of determination. (B) Changes of LCDV copies in FG and HINAE cells post LCDV infection. 0 h represented un-infected cells. Error bars represented SD. Data represented the number of LCDV copies per microgram of total DNA in the cell samples (mean ± SD; n = 3).</p

Blocking effect of anti-27.8R MAbs on 27.8R expression.

<p>The cells were pre-incubated with different concentration of anti-27.8R MAbs. Experimental groups were challenged by LCDV at a MOI of 3.0, and the cells without LCDV infection served as control groups. The cells were sampled at 48 h post infection. Error bars represented SD. Data represented the absorbance value at 405 nm (mean ± SD; n = 3) and were compared by Student’s <i>t</i> test. The asterisk represented the statistical significance (<i>p</i> < 0.05) as compared with the control group.</p

Co-localization of LCDV and 27.8R in FG and HINAE cell surface.

<p>FG cells (A) and HINAE cells (B) were exposed to LCDV at 22°C for 2 h and stained with mouse anti-27.8R MAbs and rabbit anti-LCDV serum for detection of 27.8R (green) and LCDV (red) simultaneously. The merged yellow signals (arrows) indicated the co-localization of LCDV and 27.8R protein on cell surface. Cell nuclei were counterstained in blue by DAPI. Scale bar = 20 μm. (a) and (b) were the higher magnification view of the co-localized area in FG and HINAE cells, respectively, scale bar = 5 μm.</p

Data_Sheet_1_A DNA Vaccine Encoding the VAA Gene of Vibrio anguillarum Induces a Protective Immune Response in Flounder.docx

Vibrio anguillarum is a pathogenic bacterium that infects flounder resulting in significant losses in the aquaculture industry. The VAA protein previously identified in flounder is associated with a role in immune protection within these fish. In the present study, a recombinant DNA plasmid encoding the VAA gene of V. anguillarum was constructed and its potential as a DNA vaccine, to prevent the infection of V. anguillarum in flounder fish, investigated. We verified the expression of the VAA protein both in vitro in cell lines and in vivo in flounder fish. The protective effects of pcDNA3.1-VAA (pVAA) were analyzed by determination of the percentage of sIgM+, CD4-1+, CD4-2+, CD8β+ lymphocytes, and the production of VAA-specific antibodies in flounder following their immunization with the DNA vaccine. Histopathological changes in immune related tissues, bacterial load, and relative percentage survival rates of flounder post-challenge with V. anguillarum, were all investigated to assess the efficacy of the pVAA DNA vaccine candidate. Fish intramuscularly immunized with pVAA showed a significant increase in CD4-1+, CD4-2+, and CD8β+ T lymphocytes at days 9, 11, and 14 post-vaccination, reaching peak T-cell levels at days 11 or 14 post-immunization. The percentage of sIgM+ lymphocytes reached peak levels at weeks 4–5 post-immunization. Specific anti-V. anguillarum or anti-rVAA antibodies were induced in inoculated fish at days 28–35 post-immunization. The liver of vaccinated flounder exhibited only slight histopathological changes compared with a significant pathology observed in control immunized fish. Additionally, a lower bacterial burden in the liver, spleen, and kidney were observed in pVAA protected fish in response to bacterial challenge, compared with pcDNA3.1 vector control injected fish. Moreover, the pVAA vaccine confers a relative percentage survival of 50.00% following V. anguillarum infection. In summary, this is the first study indicating an initial induction of the T lymphocyte response, followed by B lymphocyte induction of specific antibodies as a result of DNA immunization of flounder. This signifies the important potential of pVAA as a DNA vaccine candidate for the control of V. anguillarum infection.</p

Expression profiles of four genes in hemocytes of <i>F. chinensis</i> post WSSV infection.

<p>The 18S rRNA was used for normalization of PCR reactions. The bar graphs represent the relative fold changes at each time point post infection, together with error bars which represent mean ± standard deviation (n = 3). <i>Different letters</i> indicates significant difference between groups (<i>p</i><0.05). (A) SUMO, (B) heat shock protein 70, (C) arginine kinase, (D) prophenoloxidase.</p

Primers used in this work.

<p>Primers used in this work.</p

Proteomic Analysis of Differentially Expressed Proteins in <i>Fenneropenaeus chinensis</i> Hemocytes upon White Spot Syndrome Virus Infection

<div><p>To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (<i>Fenneropenaeus chinensis</i>) at 24 h post infection (hpi). Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO) 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase) and one down-regulated protein (prophenoloxidase) were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection.</p></div

The effects of silencing of <i>FcSUMO</i> and <i>FcUBC9</i>.

<p><b>(A)</b> Silencing efficiencies of dsSUMO and dsUBC9 in hemocytes of <i>F</i>. <i>chinensis</i> at 48 h post injection of dsRNA. (B) dynamic state of WSSV copies in hemocytes post WSSV infection investigated by real-time PCR; (C) Expression levels of 10 viral genes at 48 hpi, lane 1–4 represent the samples from shrimps injected with TNE+TNE, TNE+WSSV, dsGFP+WSSV, dsSUMO+WSSV and dsUBC9+WSSV respectively; (D) accumulative mortality of shrimp at 1-day interval post WSSV infection.</p