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.
Highlights
Hepatitis E virus (HEV) is a small, 27–34 nm, non-enveloped, single stranded, positive-sense,7.2 kb RNA containing virus, which enters the host mostly through oral route [1]
To gain a better understanding of the immediate changes that occur after infection, in the absence of external response modifiers present in vivo and bypass the quantitative restrictions present in proteomic tools, we investigated the host responses induced by HEV replicon when transfected alone and when transfected in Hepatitis B virus (HBV) infected Huh-7 cells established by HBV 1.3mer replicon transfection
The copy numbers of HEV in patients with dual infection was significantly higher as compared to individuals infected with HEV only (p = 0.007) (Figure S2B). [These were analyzed on patients with HBV and HEV dual infection (n = 26, Table S1) compared to patients with either HBV only (n = 19, Table S2) or HEV only infection (n = 16, Table S3)
Summary
Hepatitis E virus (HEV) is a small, 27–34 nm, non-enveloped, single stranded, positive-sense, ,7.2 kb RNA containing virus, which enters the host mostly through oral route [1]. HEV has been described to cause super-infection in chronic HBV associated liver disease patients [7,8,9,10,11,12,13,14,15] accounting for 20% of cases in regions endemic for HEV [13]. The reason for the rapid decompensation in HEV super-infection over chronic HBV infection is not understood. Both viruses are supposed to be non-cytopathic. Detailed understanding of pathobiology of HEV super-infection is indispensable in this regard
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