Background and Aims Hepatitis C virus (HCV)-induced chronic inflammation may induce oxidative stress which could compromise the repair of damaged DNA, rendering cells more susceptible to spontaneous or mutagen-induced alterations, the underlying cause of liver cirrhosis and hepatocellular carcinoma. In the current study we examined the induction of reactive oxygen species (ROS) resulting from HCV infection and evaluated its effect on the host DNA damage and repair machinery. Methods HCV infected human hepatoma cells were analyzed to determine (i) ROS, (ii) 8-oxoG and (iii) DNA glycosylases NEIL1, NEIL2, OGG1. Liver biopsies were analyzed for NEIL1. Results Human hepatoma cells infected with HCV JFH-1 showed 30–60-fold increases in ROS levels compared to uninfected cells. Levels of the oxidatively modified guanosine base 8-oxoguanine (8-oxoG) were significantly increased sixfold in the HCV-infected cells. Because DNA glycosylases are the enzymes that remove oxidized nucleotides, their expression in HCV-infected cells was analyzed. NEIL1 but not OGG1 or NEIL2 gene expression was impaired in HCV-infected cells. In accordance, we found reduced glycosylase (NEIL1-specific) activity in HCV-infected cells. The antioxidant N-acetyl cystein (NAC) efficiently reversed the NEIL1 repression by inhibiting ROS induction by HCV. NEIL1 expression was also partly restored when virus-infected cells were treated with interferon (IFN). HCV core and to a lesser extent NS3-4a and NS5A induced ROS, and downregulated NEIL1 expression. Liver biopsy specimens showed significant impairment of NEIL1 levels in HCV-infected patients with advanced liver disease compared to patients with no disease. Conclusion Collectively, the data indicate that HCV induction of ROS and perturbation of NEIL1 expression may be mechanistically involved in progression of liver disease and suggest that antioxidant and antiviral therapies can reverse these deleterious effects of HCV in part by restoring function of the DNA repair enzyme/s.

Infection with hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and end-stage liver disease worldwide. The genetics of HCV infection in humans and the disease course of chronic hepatitis C are both remarkably variable. Although the response to interferon treatment is largely dependent on HCV genotypes, whether or not a relationship exists between HCV genome variability and clinical course of hepatitis C disease still remains unknown. To more thoroughly understand HCV genome evolution over time in association with disease course, near genome-wide HCV genomes present in 9 chronically infected participants over 83 total study years were sequenced. Overall, within HCV genomes, the number of synonymous substitutions per synonymous site (dS) significantly exceeded the number of non-synonymous substitutions per site (dN). Although both dS and dN significantly increased with duration of chronic infection, there was a highly significant decrease in dN/dS ratio in HCV genomes over time. These results indicate that purifying selection acted to conserve viral protein structure despite persistence of high level of nucleotide mutagenesis inherent to HCV replication. Based on liver biopsy fibrosis scores, HCV genomes from participants with advanced fibrosis had significantly greater dS values and lower dN/dS ratios compared to participants with mild liver disease. Over time, viral genomes from participants with mild disease had significantly greater annual changes in dN, along with higher dN/dS ratios, compared to participants with advanced fibrosis. Yearly amino acid variations in the HCV p7, NS2, NS3 and NS5B genes were all significantly lower in participants with severe versus mild disease, suggesting possible pathogenic importance of protein structural conservation for these viral gene products.

The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.