Hepatitis C virus JFH-1 strain infection in chimpanzees is associated with low pathogenicity and emergence of an adaptive mutation.

UNLABELLED: The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. To assess the in vivo phenotype of the JFH-1 virus, cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. Sequencing of HCV at various times of infection indicated more substitutions in the patient serum-inoculated chimpanzee, and the higher level of sequence variations seemed to be associated with a prolonged infection in this animal. A common mutation G838R in the NS2 region emerged early in both chimpanzees. This mutation enhances viral assembly, leading to an increase in viral production in transfected or infected cells. CONCLUSION: Our study shows that the HCV JFH-1 strain causes attenuated infection and low pathogenicity in chimpanzees and is capable of adapting in vivo with a unique mutation conferring an enhanced replicative phenotype.

Diagnosis of NASH using delayed parenchymal imaging of contrast ultrasound.

BACKGROUND AND AIMS: Non alcoholic steatohepatitis (NASH) is one of the representative liver diseases in the developed countries. Diagnosis of NASH is dependent on histological findings from liver biopsy. Usefulness of contrast ultrasound with Levovist for diagnosis of NASH is described. METHODS AND MATERIALS: Clinical study: Ultrasound contrast agent, Levovist of 2.5g was injected intravenously. The liver was scanned at 5, 10, 15, 20, 30, 40, and 50min after Levovist injection in different planes using a contrast specific ultrasound mode. Changes in microbubble accumulation in the liver were evaluated. The signal intensity from regions of interest (ROI) on the contrast images was measured and accumulation and decrescence of microbubbles were estimated using the time intensity curves (TICs). The image data and TICs were evaluated by blind reviewers. Fifteen patients with NASH, 8 with alcoholic steatohepatitis (ASH), 45 with non alcoholic fatty liver (NAFL), 10 with chronic hepatitis C (CHC) and 10 healthy volunteers were studied. Animal study: Methionine-choline-deficient diet (MCDD) fed rats were used for NASH model. Correlation between microbubble accumulation and morphological and functional changes of sinusoidal endothelium and macrophage was evaluated. RESULTS: The maximum intensity of contrast ultrasound was decreased and time course decrescence was more rapid in NASH than the other groups. These changes were correlated to the degree of centrilobular and pericellular fibrosis but not to steatosis in histological study. Disturbance of microbubble accumulation was correlated with sinusoidal function rather than morphological changes such as fibrosis and parenchymatitis in the animal studies. CONCLUSIONS: The Levovist contrast study enables differential diagnosis between NASH and other diseases that provoke steatosis and fibrosis.

Differences in hepatitis C virus core protein processing among genotypes 1 and 2.

Two forms of hepatitis C virus (HCV) core protein, p23 and p21, are produced from a precursor polyprotein. Production of p21 by cleavage at the c-terminus of p23 is considered essential for viral assembly and replication. In the present experiment, an in vitro translation and transcription assays were used to examine cleavage of p21 from p23 among 19 clones isolated from patients with chronic hepatitis, including 10 infected with genotype 1, and nine infected with genotype 2. Significantly greater p21 to p23 ratios were observed among genotype 1 clones, compared to genotype 2 clones. A comparison of the amino acid sequences of these clones revealed greater production of p21 core protein among clones which contained alanine, rather than valine, at amino acid residue 189. An exploration of Hepatitis Virus Database revealed that efficient p21 production related alanine at amino acid position 189 was observed in most clones of genotype 1 and in rare clones of genotype 2. These data suggest that the efficiency of core protein production differs among genotypes depending on differences in the c-terminus amino acid sequences of their core region. This may explain differences in some of the clinical characteristics of various genotypes or clones.

Processing of hepatitis C virus core protein is regulated by its C-terminal sequence.

Polyprotein processing of plus-strand RNA viruses is important in the regulation of gene production and replication. The core protein of hepatitis C virus (HCV), constructing the viral particle, is processed from its precursor polyprotein and observed as two forms, p23 and p21. Production of p21 by cleavage at the C-terminus of p23 is considered crucial to viral assembly and replication. In this study, this processing step was compared between clones isolated from two patients with fulminant hepatitis and from five patients with chronic hepatitis by an in vitro translation assay and cell transfection assay. The p21 core protein was predominant from the clone isolated from one of the fulminant hepatitis patient (p21 core protein production was 65.98%), while p23 was abundant with clones from five chronic hepatitis patients (p21 core protein production was 7.11+/-1.62%) and clone from another fulminant hepatitis patient (p21 core protein production was 13.36%). Investigations with chimeric and mutation-introduced constructs revealed that four amino acid residues in the C-terminus of the core region are responsible for this difference. The data suggest that core protein processing is regulated by C-terminus mutations.

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.

BACKGROUND & AIMS: Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis. METHODS: A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency. RESULTS: The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/microg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/microg RNA (P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay. CONCLUSIONS: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.

Expression of survivin during liver regeneration.

Survivin functions to suppress cell death and regulate cell division, and is observed uniquely in tumor cells and developmental cells. However, the expression and regulation of survivin in non-transformed cells are not well elucidated. Therefore, we investigated the expression of survivin in a murine liver regeneration model after partial hepatectomy and intraperitoneal carbon tetrachloride (CCl(4)) injection. We found that the expression of survivin transcript and protein were markedly elevated with the onset of DNA synthesis and remained elevated during G2 and M phases during liver regeneration. In a normal mouse liver cell line, over-expression of survivin resulted in a decrease in the G0/G1 phase and an increase in the S and G2/M phases, resulting in Rb phosphorylation. These findings suggest that survivin is dramatically expressed in a cell cycle-dependent manner during liver regeneration and provide a new insight into the regulation of cell proliferation and differentiation.