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The molecular mechanisms of assembly and budding of hepatitis C virus (HCV) remain poorly understood. The budding of several enveloped viruses requires an endosomal sorting complex required for transport (ESCRT), which is part of the cellular machinery used to form multivesicular bodies (MVBs). Here, we demonstrated that Hrs, an ESCRT-0 component, is critical for the budding of HCV through the exosomal secretion pathway. Hrs depletion caused reduced exosome production, which paralleled with the decrease of HCV replication in the host cell, and that in the culture supernatant. Sucrose-density gradient separation of the culture supernatant of HCV-infected cells revealed the co-existence of HCV core proteins and the exosome marker. Furthermore, both the core protein and an envelope protein of HCV were detected in the intraluminal vesicles of MVBs. These results suggested that HCV secretion from host cells requires Hrs-dependent exosomal pathway in which the viral assembly is also involved.

Mongolia is known for its high endemicity for hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) infections among apparently healthy individuals. However, there are little or no data on the prevalence and genotype distribution of HBV, HCV, and HDV among patients with chronic liver disease in Mongolia. Therefore, serum samples obtained in 2004 from 207 patients (age, mean+/-standard deviation, 51.0+/-11.9 years) including those with chronic hepatitis (n=90), liver cirrhosis (n=41), and hepatocellular carcinoma (n=76) were tested for serological and molecular markers of HBV, HCV, and HDV infections. Of the 207 patients, 144 (69.6%), 106 (51.2%), and 117 (56.5%) tested positive for hepatitis B surface antigen (HBsAg) and/or HBV DNA, HCV RNA, and HDV RNA, respectively. Collectively, 172 patients (83.1%) were viremic for one or more of these viruses, including dual viremia of HBV/HDV (26.6%) or HBV/HCV (7.7%) and triple HBV/HCV/HDV viremia (30.0%). Of note, triple ongoing infection was significantly more frequent among patients with hepatocellular carcinoma than among those with chronic hepatitis (63.2% vs. 14.4%, P<0.0001). One hundred sixty patients (77.3%) had a history of blood transfusion and/or surgery. The distribution of HBV genotypes among the 116 HBV-viremic patients was: A (0.9%), B (0.9%), C (6.0%), D (88.8%), and C plus D (3.4%). All 117 HDV isolates were classified into genotype I. The 106 HCV RNA-positive samples were typed as genotype 1b (92.5%), 2a (0.9%), or 1b plus 2a (6.6%); mixed infection of two distinct HCV genotypes was found exclusively in the patients with hepatocellular carcinoma.

No abstract

Accumulating evidence suggests that cancer stem cells (CSC) play an important role in tumorigenicity. Epithelial cell adhesion molecule (EpCAM) is one of the markers that identifies tumor cells with high tumorigenicity. The expression of EpCAM in liver progenitor cells prompted us to investigate whether CSC could be identified in hepatocellular carcinoma (HCC) cell lines. The sorted EpCAM+ subpopulation from HCC cell lines showed a greater colony formation rate than the sorted EpCAM− subpopulation from the same cell lines, although cell proliferation was comparable between the two subpopulations. The in vivo evaluation of tumorigenicity, using supra‐immunodeficient NOD/scid/γcnull (NOG) mice, revealed that a smaller number of EpCAM+ cells (minimum 100) than EpCAM− cells was necessary for tumor formation. The bifurcated differentiation of EpCAM+ cell clones into both EpCAM+ and EpCAM− cells was obvious both in vitro and in vivo, but EpCAM− clones sustained their phenotype. These clonal analyses suggested that EpCAM+ cells may contain a multipotent cell population. Interestingly, the introduction of exogenous EpCAM into EpCAM+ clones, but not into EpCAM− clones, markedly enhanced their tumor‐forming ability, even though both transfectants expressed a similar level of EpCAM. Therefore, the difference in the tumor‐forming ability between EpCAM+ and EpCAM− cells is probably due to the intrinsic biological differences between them. Collectively, our results suggest that the EpCAM+ population is biologically quite different from the EpCAM− population in HCC cell lines, and preferentially contains a highly tumorigenic cell population with the characteristics of CSC. (Cancer Sci 2010)

No abstract

To evaluate the usefulness of detection of antibodies to hepatitis E virus (HEV) to screen for viraemic pigs, serum samples obtained from 1425 1-6-month-old pigs in Japan were tested for swine HEV RNA and IgG, IgM and IgA classes of anti-HEV antibody. Fifty-five (5 %) of the 1071 2-5-month-old pigs were positive for swine HEV RNA, but none of 218 1-month-old pigs or 136 6-month-old pigs had detectable HEV RNA. The prevalence of anti-HEV IgG among the viraemic pigs (67 %, 37/55) was similar to that among the non-viraemic pigs (55 %, 757/1370) and the prevalence of anti-HEV IgM among the viraemic pigs and non-viraemic pigs was 7 and 3 %, respectively. However, anti-HEV IgA was detected significantly more frequently among viraemic pigs than among non-viraemic pigs (55 vs 10 %, P<0?0001). These results suggest that anti-HEV IgA is more useful than anti-HEV IgM to screen for viraemic pigs.Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified as the sole member of the genus Hepevirus in the family Hepeviridae. Its genome is a single-stranded, positive-sense RNA of approximately 7?2 kb, with three partially overlapping open reading frames (ORF1, -2 and -3) (Tam et al., 1991;Huang et al., 1992;Wang et al., 2000). Although only one serotype has been recognized, extensive genomic diversity has been noted among HEV isolates, and HEV sequences have been classified into four major genotypes, 1-4 (Schlauder & Mushahwar, 2001). Transmission of HEV in developing countries occurs primarily via the faecal-oral route through contaminated water supplies . Recent studies have indicated that zoonosis is involved in the transmission of HEV, especially in industrialized countries (Meng et al., 1997(Meng et al., , 1998b(Meng et al., , 1999 Erker et al., 1999;Harrison, 1999;Meng, 2000Meng, , 2003Smith, 2001;Tei et al., 2003;Yazaki et al., 2003). Increasing lines of evidence indicate that pigs are animal reservoirs of HEV and that hepatitis E may be transmitted zoonotically from viraemic animals to humans (Meng et al., 1997;Harrison, 1999;Meng, 2000Meng, , 2003Smith, 2001;Okamoto et al., 2003;Takahashi et al., 2003a). Numerous HEV strains of genotypes 3 and 4 have been isolated from pigs in both developing and industrialized countries (Hsieh et al., 1999;Pina et al., 2000; Garkavenko et al., 2001;van der Poel et al., 2001; Arankalle et al., 2002;Huang et al., 2002a;Pei & Yoo, 2002;Wu et al., 2002;Choi et al., 2003). However, the extent of genomic variability and geographical distribution of swine HEV strains is not fully understood in Japan and there have been little or no data on the prevalence of IgM and IgA antibodies against swine HEV (anti-HEV) among domestic pigs. In the present study, we aimed to understand further the genomic heterogeneity of swine HEV strains throughout Japan and to elucidate whether detection of particular classes of anti-HEV antibodies is useful as a tool to screen for viraemic pigs.Serum samples were obtained from 1425 pigs (mean age±SD, 3?5±1?6 months, range 1-6 months) at 92 commerci...

The cellular mechanisms by which hepatitis B virus (HBV) is assembled and exported are largely undefined. Recently, it has been suggested that these steps require the multivesicular body (MVB) and the autophagic machinery. However, the mechanisms by which HBV might regulate these compartments are unclear. In this study, we have found that by activating Rab7a, HBV alters its own secretion by inducing dramatic changes in the morphology of MVB and autophagic compartments. These changes are characterized by the formation of numerous tubules that are dependent upon the increase in Rab7 activity observed in the HBV-expressing HepG2.2.15 cells compared to HepG2 cells. Interestingly, transfection-based expression of the five individual viral proteins indicated that the precore protein, which is a precursor of HBeAg, was largely responsible for the increased Rab7 activity. Finally, small interfering RNA (siRNA)-mediated depletion of Rab7 significantly increased the secretion of virions, suggesting that reduced delivery of the virus to the lysosome facilitates viral secretion. These findings provide novel evidence indicating that HBV can regulate its own secretion through an activation of the endo-lysosomal and autophagic pathway mediated by Rab7 activation.