RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

Abstract

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.

Difference in microRNA expression and editing profile of lung tissues from different pig breeds related to immune responses to HP-PRRSV

“…Growing evidence suggests that microRNA editing might involve immune responses to virus infection. During HCMV infection, edited miR-376 down-regulates the expression of HLA-E which promotes NK cell ability to eliminate the HCMV infected cells60. Further study showed that microRNA editing occurred in pre-miRNA-126, pre-miRNA-744 in Tongcheng and in pre-miRNA-27a in Landrace at 3, 5 and 7 dpi (Supplementary Table S5).…”

Hepatitis B virus molecular biology and pathogenesis

“…Recent RNA-seq analyses of HBV-expressing Huh7 cells [254] and primary rat hepatocytes [186] also detected decreased expression of GLUT2, the main hepatic glucose transporter. Investigation of the effect of fasting glucose levels on HBV replication revealed a link, albeit minor, between the metabolic state of the cell and the level of HBV replication, [245] and both gluconeogenesis and lipogenesis are under the same transcription factor control as HBV replication.…”

Recombinant Hepatitis E virus like particles can function as RNA nanocarriers

“…At various time points, post incubation (12, 24, 36, 48, 72 h; data shown for only 48 h), the cells were observed under confocal microscope using Hybrid detector (HyD) at 561 nm to check the fluorescence from RFP protein. Similar experiments were conducted for pORF2-VLPs encapsidated with 5′-methyl-G-HEV( 1 – 249 nt )- HBsAg RNA and Indirect immunofluorescence assay was performed and [ 7 ] observed under inverted fluorescent microscope (Nikon TE2000-U) for the expression of HBsAg protein. The entry and internalization of pORF2 f-VLP’s (produced from ORF2-linker EGFP protein) has been previously demonstrated by us in Huh-7 cells [ 1 ].…”