Transcriptional Profiling of Host Gene Expression in Chicken Embryo Fibroblasts Infected with Reticuloendotheliosis Virus Strain HA1101

<div><p>Reticuloendotheliosis virus (REV), a member of the Gammaretrovirus genus in the Retroviridae family, causes an immunosuppressive, oncogenic and runting-stunting syndrome in multiple avian hosts. To better understand the host interactions at the transcriptional level, microarray data analysis was performed in chicken embryo fibroblast cells at 1, 3, 5, and 7 days after infection with REV. This study identified 1,785 differentially expressed genes that were classified into several functional groups including signal transduction, immune response, biological adhesion and endocytosis. Significant differences were mainly observed in the expression of genes involved in the immune response, especially during the later post-infection time points. These results revealed that differentially expressed genes <i>IL6</i>, <i>STAT1</i>, <i>MyD88</i>, <i>TLRs</i>, <i>NF-κB</i>, <i>IRF-7</i>, and <i>ISGs</i> play important roles in the pathogenicity of REV infection. Our study is the first to use microarray analysis to investigate REV, and these findings provide insights into the underlying mechanisms of the host antiviral response and the molecular basis of viral pathogenesis.</p></div

The regulation of IFN-β production by miR-155.

<p>(a) miR-155 expression in CEF cells treated with TLR3 ligand. miR-21 expression was used as the control. (b) ELISA for the productions of IFN-α, IFN-β, IL1-β, IL6 and TNF-α in the CEF cells treated with TLR ligands. (c) Down-regulation of IFN-β in CEF cells transfected with 50–200 nM of the gga-miR-155 mimic. (d) Up-regulation of IFN-β in CEF cells transfected with 50–200 nM of the gga-miR-155 inhibitor. (e) Down-regulation of IFN-β in HD11 cells treated with 50–400 nM of the gga-miR-155 agomir. (f) Up-regulation of of IFN-β in HD11 cells treated with 50–400 nM of the gga-miR-155 antagomir.</p

Hierarchical clustering (A) and k-means clustering (B) of differentially expressed transcripts of REV infected CEFs at different post-infection time points.

<p>Expression profiles of differentially expressed transcripts with p < 0.05 at all time points and fold changes > +/- 2 at one or more time points. These significantly regulated transcripts were clustered into 9 distinct groups having similar expression response profiles over the time course of REV infection.</p

Differentially expressed genes in REV infection involved in signalling pathways.

<p>Differentially expressed genes in REV infection involved in signalling pathways.</p

The numbers of significant differentially expressed transcripts and genes.

<p>The numbers of significant differentially expressed transcripts and genes.</p

The virostatic efficacy and cytotoxicity were showed in (A) C003 and (B) C001.

<p>Cells were infected with BTV at MOI of 0.01 with or without C003 or C001 at various concentrations. At 72 h.p.i, cell viability (triangles) or cytotoxicity (circles) was measured using the CellTiter-Glo reagent. Each data point represents means and standard deviation (SD) from five replicates.</p

Effects of C003 and C052 on BTV VP6 protein expression and LC3-I/LC3-II conversion were analyzed in sample collected at different h.p.i.

<p>Cells were infected with BTV at MOI of 0.01, and treated with C003 (10 µM) or C052 (2.5 µM). VP6 expressions and LC3-I/LC3-II conversion in these samples were determined using the western blot.</p

The time-of-addition assay for C003 (A) and C052 (B).

<p>C003 at 20 and 1.76 µM, and C052 at 2.5 µM and 0.27 µM, respectively, were added to BTV infected cells at different h.p.i. as indicated, and the protection of C003 and C052 against BTV induced CPE, or cell viability, was measured using CellTiter-Glo reagent at 72 h.p.i. Each data points represented the average values and SD from of eight independent replicates.</p

Analysis of the miR-155 seed sequence in the TLR3 coding region.

<p>(a and b) The sequence logo for miR-155 was generated by Weblogo (<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>). (c) The target sites of the miR-155 sequence in the TLR3 coding regions from Homo sapiens, Gallus gallus, Danio rerio, Equus caballus, Taeniopygia guttata and Cyprinus carpio at the seed matches of the 8mer and 7mer-m8 sites. (d and e) Clustal W analysis of the miR-155 sequence with Molecular Evolutionary Genetics Analysis version 5 (MEGA5) software.</p

Effects of C003 and C052 on BTV-induced caspase-3/7 activation.

<p>BSR cells were mock infected or infected with BTV at MOI of 0.01, and treated with C003 or C052 at their EC₉₀ concentrations (A) or EC₅₀ concentrations (B). At 24, 48 and 72 h.p.i, caspase-3/-7 activities were determined using the Caspase-Glo-3/7 reagent, and presented as relative fluorescent unit (RLU). Each data points represented mean values and SD from triplicates experiments.</p