Functional Characterization of Nuclear Localization and Export Signals in Hepatitis C Virus Proteins and Their Role in the Membranous Web

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin β3 (IPO5/kap β3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.     

Virus-host interactions during hepatitis C virus entry — implications for pathogenesis and novel treatment approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.      

Differential Effect of Viral Hepatitis Infection on Mortality among Korean Maintenance Dialysis Patients: A Prospective Multicenter Cohort Study

The role of infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) in terms of survival among dialysis patients remains incompletely understood. In the present multicenter prospective cohort study, we investigated the prevalences of HBV and HCV infection among 3,321 patients receiving maintenance dialysis in Korea, and assessed the impacts of these infections on survival. All included patients underwent hepatitis B antigen (HBsAg) and HCV antibody (Ab) testing, which revealed that 236 patients (7.1%) were HBsAg-positive, and 123 patients (3.7%) were HCV Ab-positive. HBsAg-positive and HCV Ab-positive patients were matched to hepatitis virus-negative patients using a propensity score at a ratio of 1:2. The prevalences of HBV and HCV infection did not significantly differ according to dialysis modality. Linear-by-linear association analysis revealed that hepatitis B prevalence significantly increased with increasing dialysis vintage (p = 0.001), and hepatitis C prevalence tended to be higher with increasing dialysis vintage (p = 0.074). We compared the survival of HBsAg-positive and HCV Ab-positive patients to that of hepatitis virus-negative patients. After propensity score matching, cumulative survival did not differ between HBsAg-positive and HBsAg-negative patients (p = 0.37), while HCV Ab-positive patients showed significantly lower survival than HCV Ab-negative patients (p = 0.03). The main conclusions of the present study are that HBV infection prevalence increased with longer dialysis vintage, and that both HBV and HCV infections were most prevalent among patients with the longest dialysis vintage. Additionally, HCV infection among maintenance dialysis patients is associated with an increased risk of mortality.     

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPARα) signaling. Furthermore, using PPARα agonists and antagonists, we also analyzed the effect of PPARα signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.     

Characterization of the specific CD4+ T cell response against the F protein during chronic hepatitis C virus infection

Background

The hepatitis C virus (HCV) Alternate Reading Frame Protein (ARFP or F protein) presents a double-frame shift product of the HCV core gene. We and others have previously reported that the specific antibodies against the F protein could be raised in the sera of HCV chronically infected patients. However, the specific CD4⁺ T cell responses against the F protein during HCV infection and the pathological implications remained unclear. In the current study, we screened the MHC class II-presenting epitopes of the F protein through HLA-transgenic mouse models and eventually validated the specific CD4⁺ T cell responses in HCV chronically infected patients.

Methodology

DNA vaccination in HLA-DR1 and-DP4 transgenic mouse models, proliferation assay to test the F protein specific T cell response, genotyping of Chronic HCV patients and testing the F-peptide stimulated T cell response in the peripheral blood mononuclear cell (PBMC) by in vitro expansion and interferon (IFN)- γ intracellular staining.

Principal Findings

At least three peptides within HCV F protein were identified as HLA-DR or HLA-DP4 presenting epitopes by the proliferation assays in mouse models. Further study with human PBMCs evidenced the specific CD4⁺ T cell responses against HCV F protein as well in patients chronically infected with HCV.

Conclusion

The current study provided the evidence for the first time that HCV F protein could elicit specific CD4⁺ T cell response, which may provide an insight into the immunopathogenesis during HCV chronic infection.     

Hepatitis C: A mouse at the end of the tunnel

Since its discovery in 1989, researchers strive after a small animal model for Hepatitis C virus infection, so far with very limited success. A study recently published in Nature now for the first time reports the recapitulation of the complete life cycle of this virus in inbred mice with a functional adaptive immune system.Worldwide, over 130 million people are chronically infected with Hepatitis C virus (HCV). Acute infection goes along with mild and generalized symptoms, and therefore mostly remains undiagnosed; in more than half of the patients, however, the infection persists and, over the years, can cause liver damage such as fibrosis, cirrhosis or hepatocellular carcinoma.HCV is a positive strand RNA virus of the family Flaviviridae. Studies of this virus have made huge leaps forward since the implementation of efficient cell culture systems^1,2; nonetheless, our knowledge of HCV-associated pathogenesis is scarce owing to the lack of a practicable animal model. So far, chimpanzees are the only animals fully susceptible to HCV infection; however, legal, ethical, economical let alone practical reasons dictate the establishment of small animal models as an alternative. Most efforts have been put into various mouse models³, but in general, mice are resistant to HCV infection. Only individual steps of the lifecycle could be reproduced in mouse cells: expression of human variants of the entry receptors CD81 and occludin mediates virus uptake into mouse cells^4,5; selectable replicons demonstrated that HCV RNA can be replicated in mouse cells, albeit inefficiently; and assembly as well as secretion of HCV particles can be achieved in mouse hepatocytes³. In contrast, for studying the full replication cycle in living animals, systems had to be developed, in which mouse hepatic tissue was inducibly or constitutively deteriorated to allow repopulation by human hepatocytes³. This inter-species chimerism naturally required the animals to be immuno-deficient to avoid graft rejection. Nonetheless, particularly the uPA-SCID model has become very popular, especially for pre-clinical drug testing and validation and for studying passive immunization strategies against HCV infection. Still, for research on vaccine development and pathogenesis, these models are of limited to no use, as such studies require robust immune responses.The teams of Alexander Ploss and Charles Rice now report a breakthrough on the way to an immune-competent mouse model⁶. They had shown previously that adenoviral expression of human CD81 and occludin in mouse liver cells rendered fully immune-competent mice susceptible to HCV infection; however, replication was abortive⁵. In their present study, the authors take this approach two steps further: (1) by establishing mice that express the entry factors transgenically; (2) by additionally incapacitating the innate antiviral response. It had been suggested earlier that this response severely impacts HCV replication in mouse cells^7,8, and indeed, its blocking in an otherwise fully immune-competent background sufficed to increase viral replication to detectable levels in entry factor transgenic (EFT) mice. By testing knock-outs of several factors involved in innate antiviral defense, STAT1^−/− EFT mice were found to be best to support HCV replication, which was sustained for up to 11 weeks, with viral genomes detectable in both liver tissue and serum. The authors could corroborate that this viremia relied on authentic viral replication, as it could be inhibited by neutralizing antibodies or an HCV-specific antiviral compound; moreover, a drastic reduction in viral replication was observed in mice that additionally carried a knock-out of the ppia (Cyclophilin A) gene, a well-known HCV host dependency factor in humans⁹. On the down side, one has to acknowledge that the infection rate in the liver was stunningly low with only 0.4% of hepatocytes infected at a given time. In contrast, in human livers HCV antigen has been detected in around 20% of cells on average. This low rate of infection in mice is coherent with comparatively low infectivity titers of < 100 infectious units per ml mouse serum; somewhat in contrast, however, are the high RNA titers of 10⁴-10⁶ copies per ml, arguing for a high excess of non-infectious RNA-containing structures. The mechanism by which they are released into the serum of infected mice and their biophysical properties remain to be determined. Regardless of the rather sparse hepatic infection, strikingly, the authors found clear evidence for the mounting of an immune response, such as splenomegaly with increased relative frequencies of NK- and B-cells as well as infiltration of infected livers by CD8⁺ T-cells. HCV infection was apparently cleared by the T-cell response, which at late stages of infection shifted towards a memory phenotype.These results are remarkable and highlight the perspective of such an animal model possibly also for studies on (immune-mediated) pathogenesis and eventually development of T-cell activating vaccines. A few caveats, however, still need to be overcome on this front: none of the animals in the study developed a lasting, chronic infection, but rather cleared the virus after about 80 days; this might be resolved by adaptation of the virus to its new host by prolonged, serial passaging in mice. Additionally, the used transgenic mice lack STAT1, which is not only responsible for the immediate intrinsic antiviral response, but also required for signaling in response to all types of interferons (I, II and III). This deficiency for one deprives CD8⁺ T-cells of their antiviral activity through interferon-γ secretion, which has been reported to be more important in controlling HCV than direct cytolytic effects¹⁰, and, secondly, is likely to also affect the phenotypic differentiation and activation of various immune cells. One possible way to overcome the latter issue could be to use a tissue-specific knock-out of the stat1 gene, specifically targeting hepatocytes. Alternatively, a combined knock-out of different interferon effector genes with antiviral activity against HCV might still allow for HCV replication, while restoring general responsiveness to interferons.Even with the mentioned limitations, the presented strategy denotes a milestone for HCV research and is the first system that can truly be called a small animal model for HCV infection. As it does not rely on xenografts, it is not only more practicable and much cheaper than previous models, but also more robust as it is completely independent from variations of human graft donors. Albeit still in need for optimizations, finally one can see the long-awaited HCV-susceptible mouse at the end of the tunnel.     

The epidemiology and disease burden of children hospitalized for viral infections within the family Flaviviridae in China: A national cross-sectional study

BackgroundViruses of the family Flaviviridae, including Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus (YFV) and hepatitis C virus (HCV), are widely distributed worldwide. JEV, DENV and YFV belong to the genus Flavivirus, whereas HCV belongs to the genus Hepacivirus. Children’s symptoms are usually severe. As a result, rates of hospitalization due to infection with these viruses are high. The epidemiology and disease burden of hospitalized children have rarely been described in detail to date. The objective of this study was to report the general epidemiological characteristics, clinical phenotype, length of stay (LOS), burden of disease, and potential risk factors for hospitalized children infected with JEV, DENV, YFV, or HCV in Chinese pediatric hospitals.MethodologyA cross-sectional study of epidemiology and disease burden of children hospitalized for Flaviviridae virus infections between December 2015 and December 2020 in China was performed. Face sheets of discharge medical records (FSMRs) were collected from 27 tertiary children’s hospitals in the Futang Research Center of Pediatric Development and aggregated into FUTang Update medical REcords (FUTURE). Information on sociodemographic variables, clinical phenotype, and LOS as well as economic burden was included in FSMRs and compared using appropriate statistical tests.FindingsThe study described 490 children aged 0–15 years hospitalized for infections with Flaviviridae viruses. Japanese encephalitis (JE) cases are the highest, accounting for 92.65% of the total hospitalization cases caused by Flaviviridae virus infection. The incidence of JE peaked from July to October with a profile of a high proportion of severe cases (68.06%) and low mortality (0.44%). Rural children had a significantly higher incidence than urban children (91.63%). Most hospitalized dengue cases were reported in 2019 when dengue outbreaks occurred in many provinces of China, although only 14 dengue cases were collected during the study period. Yellow fever (YF) is still an imported disease in China. The hospitalizations for children with hepatitis C (HC) were not high, and mild chronic HC was the main clinical phenotype of patients. Among the four viral infections, JE had the highest disease burden (LOS and expenditure) for hospitalized children.ConclusionFirst, the present study reveals that JE remains the most serious disease due to Flaviviridae virus infection and threatens children’s health in China. Many pediatric patients have severe illnesses, but their mortality rate is lower, suggesting that existing treatment is effective. Both JEV vaccination and infection control of rural children should represent a focus of study. Second, although the dual risks of indigenous epidemics and imports of DENV still exist, the prevalence of DENV in children is generally manageable. Third, YFV currently shows no evidence of an epidemic in China. Finally, the proportion of children with chronic hepatitis C (CHC) is relatively large among hospitalized children diagnosed with HCV. Thus, early and effective intervention should be offered to children infected with HCV to ease the burden of CHC on public health.     

ICAM-1-Based Rabies Virus Vaccine Shows Increased Infection and Activation of Primary Murine B Cells In Vitro and Enhanced Antibody Titers In-Vivo

We have previously shown that live-attenuated rabies virus (RABV)-based vaccines infect and directly activate murine and human primary B cells in-vitro, which we propose can be exploited to help develop a single-dose RABV-based vaccine. Here we report on a novel approach to utilize the binding of Intracellular Adhesion Molecule-1 (ICAM-1) to its binding partner, Lymphocyte Function-associated Antigen-1 (LFA-1), on B cells to enhance B cell activation and RABV-specific antibody responses. We used a reverse genetics approach to clone, recover, and characterize a live-attenuated recombinant RABV-based vaccine expressing the murine Icam1 gene (rRABV-mICAM-1). We show that the murine ICAM-1 gene product is incorporated into virus particles, potentially exposing ICAM-1 to extracellular binding partners. While rRABV-mICAM-1 showed 10-100-fold decrease in viral titers on baby hamster kidney cells compared to the parental virus (rRABV), rRABV-mICAM-1 infected and activated primary murine B cells in-vitro more efficiently than rRABV, as indicated by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. ICAM-1 expression on the virus surface was responsible for enhanced B cell infection since pre-treating rRABV-mICAM-1 with a neutralizing anti-ICAM-1 antibody reduced B cell infection to levels observed with rRABV alone. Furthermore, 100-fold less rRABV-mICAM-1 was needed to induce antibody titers in immunized mice equivalent to antibody titers observed in rRABV-immunized mice. Of note, only 10³ focus forming units (ffu)/mouse of rRABV-mICAM-1 was needed to induce significant anti-RABV antibody titers as early as five days post-immunization. As both speed and potency of antibody responses are important in controlling human RABV infection in a post-exposure setting, these data show that expression of Icam1 from the RABV genome, which is then incorporated into the virus particle, is a promising strategy for the development of a single-dose RABV vaccine that requires only a minimum of virus.     

A new natural α-helical peptide from the venom of the scorpion Heterometrus petersii kills HCV

Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. There is no vaccine available for HCV, and almost half of patients cannot be cured using standard combination therapy. Thus, new anti-HCV strategies and drugs are urgently needed. Here, the gene encoding a new α-helical peptide, Hp1090, was screened from the venomous gland cDNA library of the scorpion Heterometrus petersii. Structural analysis showed that Hp1090 is an amphipathic α-helical peptide. In vitro HCV RNA inhibitory assays indicated that Hp1090 peptide inhibited HCV infection with an IC₅₀ of 7.62 μg/ml (5.0 μM), whereas Hp1035 peptide, showing high homology to Hp1090, exhibited no anti-HCV activity. Hp1090 acted as a viricide against HCV particles in vitro and prevented the initiation of HCV infection. Furthermore, this peptide interacted with HCV particles directly and rapidly permeabilized phospholipid membranes. Collectively, it seems that Hp1090 is virocidal for HCV in vitro, directly interacting with the viral membrane and decreasing the virus infectivity. These results suggest that Hp1090 could be considered an anti-HCV lead compound with virocidal mechanism that offers a potential therapeutic approach to HCV infection. Our work opens a new avenue for antiviral drug discovery in natural scorpion venom.     

A Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- and Serum-Derived Hepatitis C Virus

Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.     

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.     

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.     

UDP-glucuronosyltransferase 1A7 polymorphisms are associated with liver cirrhosis

Variations in the UDP-glucuronosyltransferase (UGT) 1A7 gene have been found to be related to the development of hepatocellular carcinoma (HCC). Since the pathogenesis of liver cirrhosis is not dissimilar to that of HCC, we hypothesized that UGT1A7 genetic polymorphisms may be associated with liver cirrhosis. PCR-restriction fragment length polymorphism was utilized to determine UGT for 1A7 genotypes for the 159 patients with liver cirrhosis and 263 gender/age matched controls. Simple logistic regression analysis revealed that significant risk factors for liver cirrhosis were (1) hepatitis B virus (HBV) infection, (2) hepatitis C virus (HCV) infection, (3) HBV infection plus HCV infection and (4) low-activity UGT1A7 genotypes. The results of further multivariate logistic regression confirmed these associations. Interaction of low-activity UGT1A7 genotypes and HBV (or HCV) infection produced an additive effect upon the risk for the development of liver cirrhosis [observed odds ratio (OR) (54.59) greater than the expected OR (18.05)]. UGT1A7 low/low genotype was also related to advanced liver cirrhosis (Child-Pugh classes C and/or B) (OR = 7.50, P = 0.009). This study demonstrates the novel findings that carriage of low-activity UGT1A7 genotypes represents a risk factor for the development and functional severity of liver cirrhosis.     

Factors associated with use of ultrasonography screening for hepatocellular carcinoma among hepatitis B or C carriers

Objectives: Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are important risk factors for hepatocellular carcinoma (HCC). Yet, there have been few studies on adherence to screening recommendations for groups at high risk for HCC. We assessed whether demographic factors or medical conditions affected screening participation among HBV/HCV carriers. Methods: The study population consisted of 15 565 men and women who visited the National Cancer Center, Korea between August 2002 and July 2009. A self-administered questionnaire was used to collect information on demographic characteristics, medical history, including chronic HBV and HCV infection, and health check-up history. HBV surface antigen and HCV antibody levels were measured in serum. Results: Among 781 HBV carriers, 596 (76.3%) were aware of their infection and 451 (57.8%) had ever been tested by ultrasonography. Among HCV carriers, 49 of 127 (36.6%) were aware of their infection and 61 (48.0%) had ever been tested by ultrasonography. Among HBV carriers, male sex (OR, 1.68; 95% CI, 1.22–2.31), family history of liver disease (OR, 2.04; 95% CI, 1.43–2.90), medical history of hyperlipidemia (OR, 2.70; 95% CI, 1.36–5.33), and awareness of infection status (OR, 4.30; 95% CI, 2.99–6.17) were associated with being tested. Among HCV carriers, awareness of infection (OR, 3.77; 95% CI, 1.72–8.26) was significantly associated with being tested by ultrasonography. Conclusion: Male sex, family history of liver disease, medical history of hyperlipidemia, and awareness of high risk status were associated with being tested by ultrasonography.     

Patterns of Hepatitis C Virus RNA Levels during Acute Infection: The InC3 Study

Background

Understanding the patterns of HCV RNA levels during acute hepatitis C virus (HCV) infection provides insights into immunopathogenesis and is important for vaccine design. This study evaluated patterns of HCV RNA levels and associated factors among individuals with acute infection.

Methods

Data were from an international collaboration of nine prospective cohorts of acute HCV (InC³ Study). Participants with well-characterized acute HCV infection (detected within three months post-infection and interval between the peak and subsequent HCV RNA levels≤120 days) were categorised by a priori-defined patterns of HCV RNA levels: i) spontaneous clearance, ii) partial viral control with persistence (≥1 log IU/mL decline in HCV RNA levels following peak) and iii) viral plateau with persistence (increase or <1 log IU/mL decline in HCV RNA levels following peak). Factors associated with HCV RNA patterns were assessed using multinomial logistic regression.

Results

Among 643 individuals with acute HCV, 162 with well-characterized acute HCV were identified: spontaneous clearance (32%), partial viral control with persistence (27%), and viral plateau with persistence (41%). HCV RNA levels reached a high viraemic phase within two months following infection, with higher levels in the spontaneous clearance and partial viral control groups, compared to the viral plateau group (median: 6.0, 6.2, 5.3 log IU/mL, respectively; P=0.018). In the two groups with persistence, Interferon lambda 3 (IFNL3) CC genotype was independently associated with partial viral control compared to viral plateau (adjusted odds ratio [AOR]: 2.75; 95%CI: 1.08, 7.02). In the two groups with viral control, female sex was independently associated with spontaneous clearance compared to partial viral control (AOR: 2.86; 95%CI: 1.04, 7.83).

Conclusions

Among individuals with acute HCV, a spectrum of HCV RNA patterns is evident. IFNL3 CC genotype is associated with initial viral control, while female sex is associated with ultimate spontaneous clearance.     

HLA-DQB1*03 Confers Susceptibility to Chronic Hepatitis C in Japanese: A Genome-Wide Association Study

Hepatitis C virus (HCV) establishes a chronic infection in 70-80% of infected individuals. Many researchers have examined the effect of human leukocyte antigen (HLA) on viral persistence because of its critical role in the immune response against exposure to HCV, but almost all studies have proven to be inconclusive. To identify genetic risk factors for chronic HCV infection, we analyzed 458,207 single nucleotide polymorphisms (SNPs) in 481 chronic HCV patients and 2,963 controls in a Japanese cohort. Next, we performed a replication study with an independent panel of 4,358 cases and 1,114 controls. We further confirmed the association in 1,379 cases and 25,817 controls. In the GWAS phase, we found 17 SNPs that showed suggestive association (P < 1 × 10^-5). After the first replication study, we found one intronic SNP in the HLA-DQ locus associated with chronic HCV infection, and when we combined the two studies, the association reached the level of genome-wide significance. In the second replication study, we again confirmed the association (P _combined = 3.59 × 10⁻¹⁶, odds ratio [OR] = 0.79). Subsequent analysis revealed another SNP, rs1130380, with a stronger association (OR=0.72). This nucleotide substitution causes an amino acid substitution (R55P) in the HLA-DQB1 protein specific to the DQB1*03 allele, which is common worldwide. In addition, we confirmed an association with the previously reported IFNL3-IFNL4 locus and propose that the effect of DQB1*03 on HCV persistence might be affected by the IFNL4 polymorphism. Our findings suggest that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic infection with HCV in the Japanese population and may not be independent of the IFNL4 genotype.     

Mobile genetic elements in the genome of the beneficial rhizobacterium Pseudomonas fluorescens Pf-5

Background

Three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Interestingly, CD81 is also required for Plasmodium infection. A major characteristic of tetraspanins is their ability to interact with each other and other transmembrane proteins to build tetraspanin-enriched microdomains (TEM).

Results

In our study, we describe a human hepatoma Huh-7 cell clone (Huh-7w7) which has lost CD81 expression and can be infected by HCV when human CD81 (hCD81) or mouse CD81 (mCD81) is ectopically expressed. We took advantage of these permissive cells expressing mCD81 and the previously described MT81/MT81w mAbs to analyze the role of TEM-associated CD81 in HCV infection. Importantly, MT81w antibody, which only recognizes TEM-associated mCD81, did not strongly affect HCV infection. Furthermore, cholesterol depletion, which inhibits HCV infection and reduces total cell surface expression of CD81, did not affect TEM-associated CD81 levels. In addition, sphingomyelinase treatment, which also reduces HCV infection and cell surface expression of total CD81, raised TEM-associated CD81 levels.

Conclusion

In contrast to Plasmodium infection, our data show that association of CD81 with TEM is not essential for the early steps of HCV life cycle, indicating that these two pathogens, while using the same molecules, invade their host by different mechanisms.     

Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice

The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O^Tg) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O^Tg mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.