A.R. Ciccaglione

Hepatitis C virus infection as a risk factor for hepatocellular carcinoma in patients with cirrhosis : a case-control study

OBJECTIVE To determine whether chronic hepatitis C virus (HCV) infection is an independent risk factor for hepatocellular carcinoma and whether it increases the cirrhosis-related risk for hepatocellular carcinoma. DESIGN Two pair-matched case-control studies. SETTING A referral-based hospital. PATIENTS In study I, 212 patients with hepatocellular carcinoma (197 of whom had known underlying cirrhosis) were compared with controls who had chronic nonhepatic diseases. In study II, the 197 patients with hepatocellular carcinoma and cirrhosis were compared with 197 pair-matched controls who had cirrhosis but not hepatocellular carcinoma. MEASUREMENTS Levels of antibody to HCV (anti-HCV), hepatitis B surface antigen (HBsAg), and antibody to hepatitis B core antigen (anti-HBc) were assayed, and alcohol abuse was assessed by history. MAIN RESULTS In study I, 151 patients (71%) with hepatocellular carcinoma were anti-HCV positive compared with 11 controls (5%) with chronic nonhepatic diseases (odds ratio, 42; 95% CI, 22 to 95). Multivariate analysis showed that anti-HCV was an independent risk factor for hepatocellular carcinoma (odds ratio, 69; CI, 15 to 308). The analysis also showed that HBsAg (odds ratio, 8.7; CI, 1.5 to 50) and anti-HBc (odds ratio, 4.2 (CI, 1.7 to 11) were risk factors for hepatocellular carcinoma. No statistically significant interaction was found between anti-HCV and the markers of HBV infection. In study II, 146 patients (74%) with hepatocellular carcinoma and cirrhosis were anti-HCV positive compared with 122 patients (62%) with cirrhosis alone (odds ratio, 1.8; CI, 1.1 to 2.8). Multivariate analysis confirmed that anti-HCV (odds ratio, 2.0; CI, 1.3 to 32) and HBsAg (odds ratio, 2.0; CI, 1.0 to 4.2) were independent risk factors for hepatocellular carcinoma. CONCLUSIONS Hepatitis C virus infection is a risk factor for hepatocellular carcinoma, apparently by inducing cirrhosis and, to a lesser extent, by enhancing the risk in patients with cirrhosis. Hepatitis C virus infection acts independently of HBV infection (another risk factor) and of alcohol abuse, age, or gender.

Activation of endoplasmic reticulum stress response by hepatitis C virus proteins.

Summary.Flaviviruses utilize the endoplasmic reticulum (ER) as the main site for replication and protein synthesis and cause some level of ER stress. In the present study, we evaluated the ability of HCV proteins to induce ER stress response by using a tetracycline-regulated cell line expressing a region of HCV genome containing the structural genes. In this system different levels of HCV protein expression could be obtained by varying the concentration of tetracycline in the medium. Real Time PCR and Western blotting assay demonstrated that HCV mRNA and protein levels reach a maximum value at 24–48 h and decrease at 72 h postinduction. Cell proliferation analysis indicated that HCV synthesis causes cell growth inhibition. The effect was also observed in cells expressing lower levels of HCV proteins. The expression profile of specific genes, which are markers of ER stress response, revealed the upregulation of the chaperone GRP78 and the transcription factor GADD153. Induction of GADD153 correlates with the downregulation of the antiapoptotic Bcl-2 gene suggesting that synthesis of HCV proteins may influence cell fate through the activation of ER stress signaling pathway.

Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331-383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262-291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg(339), Trp(368) and Lys(370) play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.

Reconstruction of the evolutionary dynamics of the hepatitis C virus 1b epidemic in Turkey.

Worldwide, 12.5% of the more than 170 million people infected by hepatitis C virus (HCV), live in Eastern Mediterranean countries. In Turkey, the prevalence of HCV infection ranges from 0.3% to 0.4% of the general population. We investigated the distribution of HCV subtype 1b in Turkey by analysing the NS5b viral genomic region, using a Bayesian coalescent-based framework and phylogeographical analysis to estimate the origin of the HCV 1b subtype epidemic and the genetic diversification of the virus in Turkey. The dataset consisted of 24 NS5b sequences obtained from patients chronically infected with HCV subtype 1b admitted to the different health districts of Ankara hospital plus the reference sequences for phylogenetic analysis. An independent dataset including the same 342-nt NS5b fragment from all over the world (203 sequences) was used to calibrate the evolutionary rate. Using the relaxed clock model, we estimated a mean evolutionary rate of 0.84 × 10(-3) sub/site/year (95% highest posterior density interval HPD 0.16-1.5 × 10(-3)). The results of the phylogeographical analysis suggested that the HCV epidemic probably originated in Greece during the first decade of 1900 and, a few years later (in the 1920s or 1930s), successfully spread to neighboring countries such as Turkey and Cyprus. The clustering of the majority of the Turkish strains in a single monophyletic group suggests the subsequent segregated circulation of the virus in the country during the years 1940-1999, which was probably due to unsafe medical parenteral procedures, with drug addiction playing a relatively negligible role. The Bayesian skyline plot (BSP) showed a growth in the number of effective infections between the 1940s and the 1990s, when the curve reached a plateau that still remains today, suggesting a partial success of improved transfusional policies. A coalescent-based approach to population dynamics can improve our understanding of the origin and spread of epidemics in a limited geographical area.

Evidence for the presence of autochthonous (locally acquired) cases of acute hepatitis E virus infections in Italy since the 80s

BACKGROUND Autochthonous (locally acquired) cases of acute hepatitis E virus have been recently reported in several developed countries. AIM To evidence cases, if any, and characteristics of acute hepatitis E virus infections in North-East of Italy several years ago. METHODS In 2014, stored sera of 165 nonA-nonB acute hepatitis referred to the hospital of Padua during the period 1978-1991 were tested for hepatitis C virus antibodies by EIA III and for anti-hepatitis E virus IgM by Wantai HEV IgM ELISA. Anti-hepatitis E virus IgM positive sera were tested by Real Star HEV RT-PCR kit (Altona Diagnostics, Hamburg, Germany). RESULTS Ninety-six (58.1%) sera resulted anti-HCV positive, and thus classified as acute C hepatitis. None of these subjects was anti-HEV IgM positive. Out of the 69 anti-HCV negative cases, 4 (5.8%) resulted anti-HEV IgM positive (one case hepatitis E virus-RNA positive), with an increasing trend from 2.8% during the years 1978-1984 to 9.1% during the years 1985-1991. All cases occurred in Italian patients with no travel abroad history. CONCLUSIONS There is evidence for the presence of autochthonous cases of acute hepatitis E virus infections in Italy since 80s.

Correction to: Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011–2016) Revealed by Next-Generation and Sanger Sequencing

The original version of this article unfortunately contained a mistake. The presentation of Table 1 was incorrect. The corrected table is given below. The original article has been corrected.

[428] MICROARRAY ANALYSIS OF LIVER CELLS CONTAINING A FULL-LENGHT HEPATITIS C VIRUS REPLICON

Introduction: Infection with hepatitis C virus (HCV) represents the major cause of liver disease, affecting more than 170 million individuals worldwide. Several data indicate that HCV replication can influence the progression and the severity of liver diseases by a modulation of cellular gene expression. Methods: To evaluate HCV-induced modification in the global expression profile, we compare gene expression of the human hepatoma cell line 2 1-5 carrying the full-length HCV replicon with the parental cell line Huh7 and the cured replicon cells in which the HCV replicon was removed by IFN-a treatment. Gene expression was examined using Applied Biosystems Human Genome Survey Arrays containing 3 1,700, 60-mer oligonucleotides probes representing a set of27,868 individual human genes and more than 1,000 control probes. The study was designed to analyze gene expression for three biological replicates for each cell line and two technical replicates for each biological replicate, for a total of 18 microarrays. The fold change of HCV replicon vs. parental or cured cell lines was analyzed by filtering the dataset of normalized signals (using p value 10.01 and 3) and performing ANOVA statistical analysis. PANTHER Classification system (Celera Genomics) was used to interpret gene expression data obtained comparing HCV replicon vs parental or cured cells. Alteration of specific genes was confirmed by Real-time PCR. Results and Discussion: Data obtained from this study indicated that HCV replication results in a specific and significant alteration of genes involved in cellular process such as endoplasmic reticulum (ER) and oxidative stress, apoptosis, lipid metabolism, immune defense and cell cycle which are relevant for HCV pathogenesis. Virul Hepatitis linit,