Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5' NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.     

Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines

It has been reported that hepatitis C virus (HCV) may infect and replicate in human T cells, particularly in perihepatic lymph nodes, but the extent and consequence of T-cell infection in patients is unclear. This study is conducted to characterize the parameters and functional consequences of HCV infection in T lymphocytes. By using a lymphotropic HCV strain, we showed that HCV could infect T cell lines (Molt-4 and Jurkat cells) in vitro. Both positive- and negative-strand HCV RNA were detected for several weeks after infection. Viral proteins could also be detected by immunofluorescence studies. Moreover, infectious HCV particles were produced from Molt-4 cell cultures, and could be used to infect na?ve T cell lines. HCV could also infect human primary CD4+ T cells, particularly na?ve (CD45RA+CD45RO-) CD4+ cells, in culture. The amounts of STAT-1 and phosphorylated STAT-1 proteins in the infected Molt-4 cells were significantly less than those in uninfected cultures, suggesting the possibility of defect in interferon-gamma signaling. Indeed, T-bet and STAT-1 mRNA levels after interferon-gamma stimulation in infected Molt-4 were suppressed. In conclusion, HCV could infect and transiently replicate in T cells and that HCV replication suppressed the IFN-gamma/STAT-1/T-bet signaling due to the reduction of STAT-1 and inhibition of its activation (phosphorylation).     

Hepatitis C virus infection of mononuclear cells from peripheral blood and liver infiltrates in chronically infected patients

The mechanisms underlying chronicity of hepatitis C virus (HCV) infection are poorly understood, but the importance of impaired viral clearance by the immune system has been suggested. The prevalence of HCV infection of peripheral blood mononuclear cells (PBMC) was in investigated in 34 persistently infected patients with anti-HCV (7 with liver cirrhosis, 10 with chronic active hepatitis, 5 with chronic persistent hepatitis, 4 with chronic lobular hepatitis, and 8 healthy carriers) by polymerase chain reaction (PCR). HCV infection of 116 T cell clones derived from liver infiltrating mononuclear cells obtained from 3 patients with chronic liver disease was examined using the same methods. HCV genomic sequences were found in fresh, unstimulated PBMC from 20 patients with cirrhosis, and chronic active and persistent hepatitis, but in none of the healthy carriers and only in mitogen-activated cells from 1 out of 4 patients with autoresolving chronic lobular hepatitis. Active PBMC infection was confirmed by identification of anti-genomic HCV sequences in the majority of HCV RNA-positive cells (fresh or mitogen-stimulated). A high percentage of T cell clones obtained from liver infiltrates were found to be infected by HCV. These findings suggest that HCV infection of lymphatic cells plays a role in the pathogenesis of chronically evolving liver damage. PBMC may represent a reservoir for latent infection and a site for viral multiplication. © 1995 Wiley-Liss, Inc.     

抗一HCV阳性无病毒血症患者HCV特异性免疫反应研究

目的 研究抗ＨＣＶ阳性无病毒血症患者特异的体流及细胞免疫状态。方法 对１５例抗ＨＣＶ阳性无病毒血症患者,１５例慢性ＨＣＶ持续感染者及１５例正常对照者进行研究。通过ＭＴＴ、ＬＤＨ释放法、酶联免疫等方法观察患者外周Ｔ细胞反应、自然杀伤（ＮＫ）细胞活性、细胞因子分泌及特异性体液免疫反应。结果 无病毒血症者Ｔ细胞增殖反应明显大于持续感染者及对照组,并且Ｔ细胞对核心区抗原增殖最大,ＮＳ３次之,对ＮＳ５则无明     

Hepatitis C virus infection in the family setting of patients with occult hepatitis C

Family members of patients with chronic hepatitis C virus (HCV) infection are at increased risk of HCV infection but the prevalence of HCV among family members of patients with occult HCV infection is not known. Anti‐HCV, serum HCV RNA and levels of liver enzymes were determined in 102 family members of 50 index patients with occult HCV infection and in 118 family members of 59 chronic hepatitis C index patients. HCV RNA and/or anti‐HCV were detected in 10/102 (9.8%) relatives of patients with occult HCV infection and in 4/118 (3.4%) of patients with chronic hepatitis C. Fourteen additional family members (seven were relatives of index patients with occult HCV infection) had abnormal values of liver enzymes without serological markers of HCV infection. Two of these patients (who were relatives of two index patients with occult HCV infection) underwent a liver biopsy and were diagnosed with an occult HCV infection because HCV RNA was detected in the liver cells in the absence of serological HCV markers. In conclusion, the prevalence of HCV infection among family members of patients with occult HCV infection was similar to that found among family members of patients with chronic hepatitis C. This stresses the need to adopt strategies to prevent the transmission of HCV in the family setting of patients with occult HCV infection. J. Med. Virol. 81:1198–1203, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography

Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However, flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~ 30% recovery of infectious particles. The full viability and unaltered physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development.     

Lack of evidence for the Th2 predominance in patients with chronic hepatitis C

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-gamma/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-gamma + or IL-2 +) was significantly increased in CD4 +, CD8 +, 'naive'-CD45RA + and 'memory'-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-gamma-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.     

Positive and negative strand of hepatitis C virus RNA sequences in peripheral blood mononuclear cells in patients with chronic hepatitis C: No correlation with viral genotypes 1b, 2a,and 2b

Hepatitis C virus (HCV) has many genotypes which are closely associated with the severity of chronic hepatitis and the response to antiviral therapy. Although HCV is essentially hepatotropic, several lines of evidence suggest that this virus can infect peripheral blood mononuclear cells (PBMC) in most patients with chronic HCV infection. However, the methods used previously to detect negative-strand HCV RNA have been questioned, and the PBMC tropism of different HCV genotypes remains unknown. A stringent method was used to investigate the prevalence of positive- and negative-strand HCV RNA in the PBMC of 106 patients with chronic hepatitis C and to analyze the influence of HCV genotype on the tropism of PBMC. HCV type 1b was the predominant strain in the patients. Positive-strand RNA in PBMC was detected in 83 (78%) and 40% had negative-strand RNA. The demographic and clinical features were comparable among different patients grouped by the replication status of HCV in the plasma and PBMC samples. In addition, there was no significant difference of PBMC tropism between type 1b and non-1b HCV. In summary, HCV does indeed infect actively the PBMC of chronic hepatitis C patients and such infection is not correlated to the pathogenesis of liver cell damage. Moreover, the genotype is not associated specifically with PBMC tropism of HCV. J. Med. Virol. 52:270–274, 1997. © 1997 Wiley-Liss, Inc.     

Suggested role of the Golgi apparatus and endoplasmic reticulum for crucial sites of hepatitis C virus replication in human lymphoblastoid cells infected in vitro

Iacovacci et al. [(1997a) Research in Virology 148:147-151] described that the euploid diploid cells, of the normal human bone marrow-derived lymphoblastoid B-cell line TO.FE., are susceptible to hepatitis C virus (HCV) infection and support long term virus production. Transmission electron microscopy described some steps of HCV replication cycle in this in vitro infected cellular system [Serafino et al. (1997) Research in Virology 148:153-159]. In the present study, in order to identify the intracellular sites involved in HCV replication, the ultrastructural changes associated with infection in TO.FE. cells were correlated with the subcellular localisation of structural and nonstructural viral proteins. Transmission electron microscopy and confocal microscopy data indicate that these viral proteins appeared located in the Golgi apparatus and endoplasmic reticulum, suggesting an active involvement of these compartments in viral assembly and morphogenesis. Furthermore, transmission and scanning electron microscopic observations on cultures infected chronically support the hypothesis that these cellular compartments may serve as starting sites of the morphological changes associated to viral infection and replication, leading to cell-cell fusion, syncytia formation, and finally lysis of infected cells and virus release.     

Cell culture-adaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity

Remarkable advances have been made through recent demonstrations of hepatitis C virus (HCV) replication in cultured cells transfected with in vitro synthesized viral genome RNA. From the HCV JFH1 (genotype 2a) subcultured successively in Huh-7 cells we have identified several missense mutations near the junction of NS5A and NS5B genes. Reverse genetic analysis indicated that two mutations in the N-terminal region of NS5B replicase caused delayed viral RNA replication and protein expression in the early stage of infection. However, the mutant viruses showed significantly alleviated effects on cell growth inhibition, proteolysis of viral proteins, apoptotic DNA cleavage, and induction of antiviral responses, giving rise to a 100-fold higher titer compared to the parental JFH1 virus in a more extended time period. These results suggested that delayed replication and reduced cytotoxicity can be characteristic features of cell culture-adaptive mutants with enhanced infectivity.     

Splenic large B-cell lymphoma in patients with hepatitis C virus infection

Hepatitis virus infection, especially type C (hepatitis C virus [HCV]), has been suggested to be one of the important pathogenetic factors for low- and high-grade B-cell lymphoma, including splenic marginal zone lymphoma (SMZL), in southern Europe. Here, we analyzed the incidences of HCV and hepatitis B virus (HBV) infections, and the clinicopathologic features in 29 cases of splenic diffuse large B-cell lymphoma (DLBCL), 10 SMZL, 3 splenic mantle cell lymphoma, 1 hairy cell leukemia, 13 B-chronic lymphocytic leukemia, and 12 hepatosplenic T-cell and natural killer cell lymphoma. Fifteen (51.7%) splenic DLBCL cases were HCV antibody-positive, and another 6 (20.7%) had the HBsAg. The incidence of each was significantly (P < .01) higher than those of HCV (9.3%) and HBV (1.9%) infections in 54 node-based DLBCL cases. Four examined HCV-positive DLBCL cases showed no type II cryoglobulinemia. HCV RNA was detected in fresh tumor tissues from 6 of 7 examined DLBCL cases, and HBV DNA was present in another 2, as evaluated by real-time polymerase chain reaction. Immunohistologically, tumor cells in 5 of 7 examined DLBCL cases showed intracytoplasmic reactions for HCV NS3 and E2 proteins and the viral receptor CD81. Of 6 cases, 2 showed an intranuclear reaction for the HBV surface protein. By Southern blot analysis, no rearrangement of the Bcl2 gene was detected in the tumor tissue of 7 HCV-positive DLBCL cases. For the other types of malignant lymphoma, 1 case each of SMZL (10%) and hepatosplenic T-cell and natural killer cell lymphoma (8.3%) showed HCV infection. In conclusion, persistent human hepatitis virus infections, especially HCV, may play an important role in the tumorigenesis of splenic DLBCL in Japan.     

Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22''s targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.     

Is replication of hepatitis C virus a marker of activity of infectious process? (Findings of polymerase chain reaction and morphological analysis of liver biopsy specimens)

Comparative study of replication markers of hepatitis C virus in various biological substrates and evaluation of activity of chronic HCV infection in liver biopsy specimens showed that replication of hepatitis C virus and the number of HCV-infected hepatocytes did not promote liver damage in chronic hepatitis C. These findings indicate that the expression of antiviral reactions by liver parenchyma cells plays the key role in the morphogenesis of HCV infection.     

Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions

We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.     

Trans-complemented hepatitis C virus particles as a versatile tool for study of virus assembly and infection

In this study, we compared the entry processes of trans-complemented hepatitis C virus particles (HCVtcp), cell culture-produced HCV (HCVcc) and HCV pseudoparticles (HCVpp). Anti-CD81 antibody reduced the entry of HCVtcp and HCVcc to almost background levels, and that of HCVpp by approximately 50%. Apolipoprotein E-dependent infection was observed with HCVtcp and HCVcc, but not with HCVpp, suggesting that the HCVtcp system is more relevant as a model of HCV infection than HCVpp. We improved the productivity of HCVtcp by introducing adapted mutations and by deleting sequences not required for replication from the subgenomic replicon construct. Furthermore, blind passage of the HCVtcp in packaging cells resulted in a novel mutation in the NS3 region, N1586D, which contributed to assembly of infectious virus. These results demonstrate that our plasmid-based system for efficient production of HCVtcp is beneficial for studying HCV life cycles, particularly in viral assembly and infection.     

Immunohistochemical expression of CD95 (Fas), c-myc and epidermal growth factor receptor in hepatitis C virus infection, cirrhotic liver disease and hepatocellular carcinoma

Gene product expression in normal and chronic hepatitis C virus infection was determined in an attempt to improve our understanding of the molecular events leading to the development of cirrhosis and liver carcinoma. Activation of CD95 (Fas) causes apoptosis of cells and liver failure in mice and has been associated with human liver disorders. c-myc is involved in cell proliferation and EGFR in regeneration of cells. The material of the current study included 50 cases of chronic hepatitis C (CHC) (and negative hepatitis B virus infection), 29 cases of liver cirrhosis and HCV (LC), and 19 cases of hepatocellular carcinoma and HCV (HCC) admitted to the Theodor Bilharz Research Institute (TBRI) during the years 2003-2004. Ten wedge liver biopsies - taken during laparoscopic cholecystectomy - were included in the study as normal controls. Laboratory investigations, including liver function tests, serological markers for viral hepatitis and serum alpha fetoprotein level (alpha-FP), were determined for all cases. Histopathological study and immunohistochemistry using monoclonal antibodies for CD95, c-myc and EGFR were also done. In CHC cases, the histological activity index (HAI) revealed more expression of Fas antigen in liver tissues with active inflammation than in those without active inflammation (p < 0.01). EGFR and c-myc act synergistically in liver tumorigenesis. Upregulation of Fas in chronic hepatitis C infection and of c-myc & EGFR in malignant transformation was concluded from this study. c-myc expression may obstruct the induction of apoptosis of HCC cells and lead to uncontrolled cell growth.