7 research outputs found
Identification of Multipath Genes Differentially Expressed in Pathway-Targeted Microarrays in Zebrafish Infected and Surviving Spring Viremia Carp Virus (SVCV) Suggest Preventive Drug Candidates
<div><p>Spring viremia carp virus (SVCV) is a rhabdovirus seasonally affecting warm-water cyprinid fish farming causing high impacts in worldwide economy. Because of the lack of effective preventive treatments, the identification of multipath genes involved in SVCV infection might be an alternative to explore the possibilities of using drugs for seasonal prevention of this fish disease. Because the zebrafish (<i>Danio rerio</i>) is a cyprinid susceptible to SVCV and their genetics and genome sequence are well advanced, it has been chosen as a model for SVCV infections. We have used newly designed pathway-targeted microarrays 3-4-fold enriched for immune/infection functional-relevant probes by using zebrafish orthologous to human genes from selected pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative analysis of differential expression of genes through 20 pathways in 2-day exposed or 30-day survivors of SVCV infection allowed the identification of 16 multipath genes common to more than 6 pathways. In addition, receptors (Toll-like, B-cell, T-cell, RIG1-like) as well as viral RNA infection pathways were identified as the most important human-like pathways targeted by SVCV infection. Furthermore, by using bioinformatic tools to compare the promoter sequences corresponding to up and downregulated multipath gene groups, we identified putative common transcription factors which might be controlling such responses in a coordinated manner. Possible drug candidates to be tested in fish, can be identified now through search of data bases among those associated with the human orthologous to the zebrafish multipath genes. With the use of pathway-targeted microarrays, we identified some of the most important genes and transcription factors which might be implicated in viral shutoff and/or host survival responses after SVCV infection. These results could contribute to develop novel drug-based prevention methods and consolidate the zebrafish/SVCV as a model for vertebrate viral diseases. </p> </div
Microarray hybridization and RTqPCR fold comparison of differentially expressed multipath genes.
<p>Microarray folds of the differentially expressed multipath genes from Table 1 were compared with the corresponding folds obtained by RTqPCR as described in Methods. To increase clarity the genes were distributed in three groups: <b>A</b>, <b>B</b> and <b>C</b>. Points inside the shadowed ellipses include most of the data. Logarithmic scales were used to best compare all data because of the wide relative differences between microarray and RTqPCR fold values. ○, Mean folds from head kidney and spleen from 2-day infected zebrafish. ●, Mean folds from head kidney and spleen from 30-day survivor zebrafish. Arrows indicate the direction of the fold changes from 2- to 30-days by both microarray and RTqPCR estimations. The <i>mapk10</i>, a multipath gene whose fold did not changed from 2- to 30-days, was included as a control (Figure 4C).</p
VENN diagram between genome-wide and targeted microarrays and comparison of fluorescence intensities obtained by hybridization to targeted microarrays of transcripts from 2- and 30-days after SVCV infection with those obtained from non-infected zebrafish controls.
<p><b>A</b>) A VENN diagram was constructed by comparing unique accession numbers between genome-wide zebrafish vs2 ID019161 microarray of Agilent (43803 probes, 37464 accession numbers) and targeted microarray zfin ID041401 (11586 probes, 8636 unique accession numbers: 2286 and 6350 pathway and keyword sections, respectively). The online software from BioInfoRx (<a href="http://apps.bioinforx.com" target="_blank">http://apps.bioinforx.com</a>) was used. <b>A1</b>, wide-genome zebrafish vs 2 ID019161. <b>A2</b>, pathway section of the targeted microarray zfin ID041401. A3, keyword section of the targeted microarray zfin ID041401. <b>B</b>, <b>C</b>) The zfin ID041401 was used to estimate transcript levels in pooled head kidney and spleen from SVCV-infected and non-infected zebrafish after 2-days (<b>B</b>) or 30-days (<b>C</b>). The Figure shows the range of mean fluorescences obtained from different experiments (6 fish per experiment, n=3 for non-infected zebrafish and for SVCV infected zebrafish after 2-days and n=2 for SVCV infected zebrafish after 30-days). A white straight line shows fold = 1.</p
“Toll-like receptor signaling pathway” of zebrafish genes after SVCV infection.
<p>The gene boxes containing the zebrafish gene short names in <i>italics</i> and their relationships were obtained from the corresponding human KEGG-pathway. All those genes appearing in the Figure were assayed for transcript expression. Capital letters, inputs (black) and outputs (blue) of the pathway. Vertical parallel lines, cellular membranes. Continuous black arrows, activation between gene products. Discontinuous black arrows, inhibition between gene products. Gray form, main pathway. Folds were calculated as described in methods by the formula, fluorescence value 2- or 30-days after infection / fluorescence mean value in non-infected controls. Red <i>italic</i> letters, downregulated transcripts with folds <0.5. Orange <i>italic</i> letters, downregulated transcripts with folds <0.66. Bright green <i>italic</i> letters, upregulated transcripts with folds >2. Dark green <i>italic</i> letters, upregulated transcripts with folds >1.5. Black <i>italic</i> letters, not differentially expressed. Gene short names surrounded by an square, multipath genes. Gray form, downstream pathway beginning with cell membrane toll-like receptors (tlr1, tlr2, tlr4, tlr5b and tlr6). Ovoid brown forms, <i>tlrs</i>. Gene <i>italic</i> letters were colored as follows: red <0.5, orange <0.66, dark green >1.5 and bright green >2 folds. <b>A</b>, 2-days after SVCV infection. <b>B</b>, 30-days after SVCV infection (survivors).</p
Immunofluorescence microscopy of ZF4 cell monolayers after SVCV infection reveals synthesis of <i>nfkb2</i> protein and its nuclear translocation.
<p>ZF4 cell monolayers were either mock infected (<b>A</b>) or infected with 0.001 SVCV per cell (<b>B</b>). Two days later the cell monolayers were stained with anti-human <i>nfkb/p65</i> -TRIC-labeled secondary antibodies and with DAPI (nuclear staining) as indicated in methods. <b>1</b>, Fluorescence with anti-<i>nfkb/p65</i> -TRIC- labeled antibodies. <b>2</b>, Fluorescence with DAPI. <b>3</b>, Merged fields 1 and 2.</p
Differential expression of Mx, C-reactive proteins (CRP), high mobility group (HMG), and antimicrobial peptides (AM) 2- and 30- days after SVCV infection.
<p>The data were obtained from the keyword section of the targeted microarray. After normalization, means and their standard deviations were represented. Short gene names (<i>italics</i>) with differential expression folds > 2 (black names) or < 0.5 (red names) are to the right of the corresponding bars. The same short gene names were used for different bars because they corresponded to different probes per gene. Black bars, similar folds at 2- and 30-days. +, increased folds in black. -, decreased folds in red. <b>A</b>) 2-days after infection. <b>B</b>) 30-days after infection.</p
Number of multipath genes per KEGG pathway.
<div><p>The number of multipath genes having differential expression folds <0.66 or > 1.5 (differentially expressed multipath genes of Table 1) in each of the 20 studied KEGG pathways, were represented.</p>
<p>Black bar, Toll-like receptor signaling pathway. Hatched bars, rest of the pathways.</p></div