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

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

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

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

MOESM1 of Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus)

Additional file 1: Table S1. Bacterial strains, plasmids and primers used in this study. Figure S1. Mass spectrographic analysis of the G protein expressed by L. lactis NZ9000. (A) The amino acid sequence of HIRRV-G protein. Eight matched peptides were underlined and two of the best matched peptides were labeled in bold. (B) Fingerprints of the two best matched peptides. (C) The matched protein information of mass spectrometric analysis

Quantitative real-time RT-PCR analysis of tissue distribution of <i>FcSUMO</i> and <i>FcUBC9</i> transcripts in healthy <i>F</i>. <i>chinensis</i>.

<p>18S rRNA served as an internal control. Transcription level of the genes in muscle was normalized to 1. The bar graphs represent the relative fold changes of each tissue, 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) UBC9.</p