ABSTRACT: Prevalence of hepatitis C virus subgenotypes 1a and 1b in Japanese patients: ultra-deep sequencing analysis of HCV NS5B genotype-specific region
Project description:Prevalence of hepatitis C virus subgenotypes 1a and 1b in Japanese patients: ultra-deep sequencing analysis of HCV NS5B genotype-specific region
Project description:Hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, belongs to the Flaviviridæ family and contains a single-strand positive-sense RNA genome, which upon virus entry and uncoating, functions as mRNAs and thus can be directly translated into proteins by host cell machinery. To date the HCV origin remains unclear and HCV life cycle and pathogenesis are not enlightened processes due to the absence of HCV efficient cell cultures systems or animals models. Here we show that rabbit and hare HCV-like viruses, RHCV and HHCV respectively, are formed after the inoculation of genomic DNA in Madin-Darby bovine kidney cell line cultures. RHCV is closely related to the HCV-1a/HCV-1b genotypes and HHCV is more closely related to the HCV-1b genotype. These findings could contribute to the understanding of HCV origin as well as clarify the virus life cycle, pathogenesis, evolution and diversity.
Project description:Hepatitis C Virus (HCV) has a extremely narrow host cell tropism and robustly infects only very few cell lines, most importantly the human hepatoma cell line Huh7. This cell line was isolated from a 57-year old Japanese male with fulminant hepatitis. Different subclones and passages of the Huh7 cell line show up to 1000-fold differences in HCV replication efficiency (permissiveness). In this experiment, we sought to identify factors responsible for these differences by correlating gene expression from eight different uninfected Huh7 variants with their respective HCV permissiveness. HCV replication efficiency was determined using electroporation of a subgenomic luciferase reporter replicon (genotype 1b, "con1/ET") and measuring luciferase activity at 48h post transfection normalized to the input value at 4h p.t.. "Relative permissiveness" of cell lines corresponds to their replication efficiency, normalized to the efficiency in the lowest permissive cells (Huh7 p13 and p28).
Project description:Primary human hepatocytes (PHHs) are a liver-specific cell subtype, and we have shown that these cells respond in a unique manner to the introduction of hepatitis C viral RNA (HCV vRNA) derived from different genotypes of the virus. We used microarray to analyze the transcriptional differences between the PHHs exposed to the different genotypes of HCV to further shed light on their differential effects on HCV innate immune responses in vitro HCV vRNA from either genotype 3a HCV or genotype 1a HCV was introduced into the PHH cells for 8 hours. Total RNA was then harvested to determine transcriptional differences.
Project description:Equine hepacivirus (EqHV) is phylogenetically the closest relative of hepatitis C virus (HCV) and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease, and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. Approach & Results: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same, and subsequently a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred non-sterilizing immunity resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV specific T cells were identified. Additionally, an interferon stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA, miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. Conclusions: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.
Project description:A miRNA microarray was performed from HCV infected patient serum samples of bothe genotype 1b and genotype 3a, which are prevalent in India, with the aim of identifying a set of miRNAs which are uniquely differentially expressed during HCV infection. miR-320c, miR-483-5p, miR-134 and miR-198 were found to be upregulated in the patient samples as compared to the controls and are currenty being validated.
Project description:Equine hepacivirus (EqHV) is the closest genetic relative of hepatitis C virus (HCV) and shares features of genome organization, hepatotropism, persistent infection, and the ability to cause liver disease. As such, EqHV studies are important both in order to understand equine liver disease, and as an outbred animal model for HCV pathogenesis and immune responses. Here, we characterize the natural history and immune response to EqHV infection. Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and challenge inoculated with the same, and subsequently a divergent EqHV inoculum. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Resolving horses developed non-sterilizing immunity resulting in short duration of infection upon challenge. Unlike those observed in acutely HCV-infected patients, peripheral blood mononuclear cell responses in horses were minimal, although EqHV specific T-cells were identified. In contrast, an interferon stimulated gene signature was detected in the liver during EqHV infection, which is similar to acute HCV in humans. EqHV, similarly to HCV, is stimulated by direct binding of the liver-specific microRNA, miR-122. Surprisingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. Conclusion: EqHV infection in horses typically has an acute resolving course, and the immune response attenuates subsequent infections lasting for at least a year. This could have important implications to achieve the first goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after challenge.
Project description:Human hepatocyte chimeric mice were prepared and treated with hepatitis C virus (HCV) and/or interferon-alpha (IFN-α). To analyze the changes in gene expression, cDNA microarray analysis was performed with the collected human hepatocytes from the chimeric mouse livers. We consider that these results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy. A total of 15 human hepatocyte chimeric mice were prepared and divided into four experimental groups. Mice in group A were neither infected with HCV nor treated with IFN. Mice in group B were administered IFN-α 6 h before sacrifice but were not infected with HCV. The mice in groups C and D were both inoculated via the mouse tail vein with human serum containing HCV genotype 1b particles. At 8 weeks after inoculation, the mice in group D were administered IFN-α 6 h before sacrifice, but the mice in group C were not treated with IFN-α. The human hepatocytes in the mouse livers were collected after sacrifice and subjected to microarray analysis. After purification and reverse transcription of total RNA, cDNA was hybridized on Affymetrix GeneChip Human Gene U133 Plus 2.0 Arrays.