Rhythm and Refrain: In Between Philosophy and Arts (2016)

<p>Pictorial representation of few important host defense response and apoptosis related genes that were differentially expressed (up regulated [green]; down regulated [red]) in HEV replicon transfected cell cultures compared to pcDNA3 only control.</p

Prospects and progress of DNA vaccines for treating hepatitis B

The hepatitis B virus (HBV) is a global cause of liver disease. The preventive HBV vaccine has effectively reduced the disease burden. However, an estimated 340 million chronic HBV cases are in need of treatment. Current standard therapy for chronic HBV blocks reversed transcription. As this therapy blocks viral maturation and not viral protein expression, any immune inhibition exerted by these proteins will remain throughout therapy. This may help to explain why these drugs rarely induce off-therapy responses. Albeit some restoration of immune function occurs during therapy, this is clearly insufficient to control replication. Central questions when considering therapeutic DNA vaccination as an addition to blocking virus production are as follows: what does one hope to achieve? What do we think is wrong and how can the vaccination correct this? We here discuss different scenarios with respect to the lack of success of tested DNA vaccines, and suggest strategies for improvement

RNA-seq based transcriptome analysis of hepatitis E virus (HEV) and hepatitis B virus (HBV) replicon transfected Huh-7 cells.

Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV

Selected genes for gene expression validation by real-time PCR.

<p>Selected genes for gene expression validation by real-time PCR.</p

ERCC spike-in mixes QAQC.

<p>ERCC spike-in mixes QAQC.</p

Differentially expressed genes in HEV, HBV and HEV+HBV transfected cell cultures compared to pcDNA3 vector-only control.

<p>The differential gene expression by Anova for each comparison, HEV vs. pcDNA3, HBV vs. pcDNA3 and HBV+HEV vs. pcDNA3 resulted in (A) 306, (B) 408 and (C) 461 genes with more than +/−2 fold change in expression (up regulated genes are marked in blue and down regulated genes are marked in red) and p value <0.05, respectively. (D) The Venn diagram shows common genes in the three groups.</p

Enriched Gene ontology annotation of differentially expressed genes in HEV, HBV and HBV+HEV transfected cells.

<p>XY bar charts represent GO type (A, B and C) and functions (D, E and F) on x-axis against mean enrichment scores on y-axis. GO types include biological process, cellular component and molecular function represented in red, blue and green colors, respectively. GO enrichment of the differentially expressed genes showed a higher enrichment for HBV+HEV (C) group followed by HEV (A) and HBV (B).</p

Relative quantification of differential gene expression by real-time RT PCR for validation.

<p>The XY plots represent the normalized log fold expression of genes obtained by real-time PCR as compared to pcDNA3 control (baseline represents expression in pcDNA3 control).</p

Post-alignment QAQC using Tophat2 aligner.

<p>Post-alignment QAQC using Tophat2 aligner.</p

Graphical representation of host defense and survival functions derived by GO enrichment of differentially expressed genes in HBV transfected cell cultures compared to pcDNA3 control.

<p>Bars represent number of genes in the list present in each functional category.</p