[Personalized treatment of viral hepatitis of the present and the future : Hepatitis B, C, delta, and E]. / Maßgeschneiderte Therapie der Virushepatitis der Gegenwart und Zukunft : Hepatitis B, C, Delta und E.

Precision medicine is also possible for infectious diseases as shown for the treatment of chronic viral hepatitis, especially if different options are available. In hepatitis B virus (HBV) infection, treatment indication as well as the choice of treatment and the decisions to stop treatment are based on viral markers and alanine aminotransferase (ALT) level. Future therapies for HBV infection aiming for functional cure or even virus elimination may be even more personalized and have to take into account the immune status of a given patient. Such treatment modalities might also increase the chance for successful treatment of chronic hepatitis delta where treatment options are still very limited. Some new therapeutic concepts targeting host receptors or host enzymes are promising, but may require individualized approaches. Chronic hepatitis C is a good example for precision medicine based on viral and host factors. However, the main reason for individualized direct-acting antiviral (DAA) treatment is to save costs. As DAAs are effective in more than 95% of patients, elimination of HCV seems to be possible at the level of a given country or even on a global scale. However, owing to high reinfection rates in high-risk groups and limited availability of antiviral therapy in many high endemic countries, it must still be decided whether an HCV vaccine or pre-exposure prophylaxis is required to achieve this goal. Hepatitis E is an emerging topic as this is the most frequent acute hepatitis virus infection. It can result in a chronic infection in immunosuppressed individuals. Treatment options are still limited and individualized management is based on tailoring immunosuppressive therapy and therapy with ribavirin. Thus, personalized therapy of hepatitis E virus infection is still limited.

Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells.

The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

Thermostability of seven hepatitis C virus genotypes in vitro and in vivo.

Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28 days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2 days was observed at 37 °C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures.

Virucidal activity of oral, hand, and surface disinfectants against respiratory syncytial virus.

BACKGROUND: Respiratory syncytial virus (RSV) is known as a major cause of respiratory tract infection in adults and children. Human-to-human transmission occurs via droplets as well as direct and indirect contact (e.g. contaminated surfaces or hands of medical staff). Therefore, applicable hygiene measures and knowledge about viral inactivation are of utmost importance. AIM: To elucidate the disinfection profile of RSV. METHODS: The study evaluated the virucidal efficacy of oral rinses specifically designed for children, World Health Organization (WHO)-recommended hand-rub formulations, and ethanol, as well as 2-propanol against RSV in a quantitative suspension test (EN14476). The stability of RSV on stainless steel discs was assessed and its inactivation by different surface disinfectants (EN16777) investigated. FINDINGS: All tested oral rinses except one reduced infectious viral titres to the lower limit of quantification. The two WHO-recommended hand-rub formulations as well as 30% ethanol and 2-propanol completely abolished the detection of infectious virus. Infectious RSV was recovered after several days on stainless steel discs. However, RSV was efficiently inactivated by all tested surface disinfectants based on alcohol, aldehyde, or hydrogen peroxide. CONCLUSION: Oral rinses, all tested hand-rub formulations as well as surface inactivation reagents were sufficient for RSV inactivation in vitro.

Hepatitis C virus enters human peripheral neuroblastoma cells - evidence for extra-hepatic cells sustaining hepatitis C virus penetration.

Patients with chronic hepatitis C virus (HCV) infection show an increased incidence of nervous system disorders such as chronic fatigue syndrome, depression and cognitive dysfunction. It is unclear whether this is because of HCV replication in the brain and in peripheral neuronal cells or to more indirect effects of HCV infection on the central or peripheral nervous system. The aim of this study was to investigate whether cells originating from these tissues are permissive for HCV cell entry, RNA replication and virus assembly. Among eight cell lines analysed, the human peripheral neuroblastoma cell line SKNMC expressed all HCV entry factors and was efficiently infected with HCV pseudoparticles (HCVpp) independent of the HCV genotype. All remaining cell types including human neuroblastoma and glioblastoma cell lines and microglial cells lacked expression of at least one host factor essential for HCV entry. When transfected with HCV luciferase reporter virus RNA, inoculated with HCV reporter viruses or challenged with high-titre cell culture-derived HCV, none of these cells supported detectable HCV RNA replication. Thus, in conclusion, this comprehensive screening did not reveal evidence directly strengthening the notion that HCV enters and replicates in the central nervous system. However, productive viral entry into the peripheral neuroblastoma cell line SKNMC indicates that HCV may penetrate into certain nonhepatic cell types which may serve as viral reservoirs and could modulate viral pathogenesis.

Long-term follow-up of successful hepatitis C virus therapy: waning immune responses and disappearance of liver disease are consistent with cure.

BACKGROUND: A sustained viral response (SVR) after interferon-based therapy of chronic hepatitis C virus (HCV) infection is regarded to represent a cure. Previous studies have used different markers to clarify whether an SVR truly represents a cure, but no study has combined a clinical work-up with highly sensitive HCV RNA detection, and the determination of immune responses. AIM: To determine clinical, histological, virological and immunological markers 5-20 years after SVR. METHODS: In 54 patients, liver biochemistry, histology and elastography were evaluated. Liver biopsies, plasma and peripheral blood mononuclear cells (PBMCs) were tested for minute amounts of HCV RNA. HCV-specific T-cell responses were monitored by ELISpot and pentamer staining, and humoral responses by measuring HCV nonstructural (NS)3-specific antibodies and virus neutralisation. RESULTS: Liver disease regressed significantly in all patients, and 51 were HCV RNA-negative in all tissues tested. There was an inverse association between liver disease, HCV-specific T-cell responses and HCV antibody levels with time from SVR, supporting that the virus had been cleared. The three patients, who all lacked signs of liver disease, had HCV RNA in PBMCs 5-9 years after SVR. All three had HCV-specific T cells and NS3 antibodies, but no cross-neutralising antibodies. CONCLUSIONS: Our combined data confirm that a SVR corresponds to a long-term clinical cure. The waning immune responses support the disappearance of the antigenic stimulus. Transient HCV RNA traces may be detected in some patients up to 9 years after SVR, but no marker associates this with an increased risk for liver disease.

The hepatitis C virus replicon system and its application to molecular studies.

The hepatitis C virus (HCV) was identified as the major causative agent of post-transfusion and sporadic non-A, non-B hepatitis. Approximately 170 million individuals worldwide are afflicted with this infection that in most cases becomes persistent. The clinical outcomes are varied, ranging from an apparently healthy carrier state to liver cirrhosis and even hepatocellular carcinoma. Thus far, no vaccine is available and antiviral treatment is insufficient with only approximately 40% of patients developing a long-term sustained response. These deficits underscore the need for more effective therapies but their development has been severely hampered by the lack of an efficient cell culture system. This impediment has recently been overcome by the development of subgenomic HCV RNA molecules that replicate autonomously in transfected cells. The high level of replication of this system opens new avenues for molecular studies of various aspects of the HCV life-cycle as well as for the development of antiviral drugs.

Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea.

The consumption of green tea (Camellia sinensis) has been shown to have many physiological and pharmacological health benefits. In the past two decades several studies have reported that epigallocatechin-3-gallate (EGCG), the main constituent of green tea, has anti-infective properties. Antiviral activities of EGCG with different modes of action have been demonstrated on diverse families of viruses, such as Retroviridae, Orthomyxoviridae and Flaviviridae and include important human pathogens like human immunodeficiency virus, influenza A virus and the hepatitis C virus. Furthermore, the molecule interferes with the replication cycle of DNA viruses like hepatitis B virus, herpes simplex virus and adenovirus. Most of these studies demonstrated antiviral properties within physiological concentrations of EGCG in vitro. In contrast, the minimum inhibitory concentrations against bacteria were 10-100-fold higher. Nevertheless, the antibacterial effects of EGCG alone and in combination with different antibiotics have been intensively analysed against a number of bacteria including multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus or Stenotrophomonas maltophilia. Furthermore, the catechin EGCG has antifungal activity against human-pathogenic yeasts like Candida albicans. Although the mechanistic effects of EGCG are not fully understood, there are results indicating that EGCG binds to lipid membranes and affects the folic acid metabolism of bacteria and fungi by inhibiting the cytoplasmic enzyme dihydrofolate reductase. This review summarizes the current knowledge and future perspectives on the antibacterial, antifungal and antiviral effects of the green tea constituent EGCG.

A century of international drug control.

A century ago, the Chinese opium epidemic spurred international action on drug control as policymakers realized that the problem was too complex for any one country to tackle in isolation. Starting with the International Opium Commission (Shanghai, 1909), Governments over time established an international consensus on the need for the regulation of psychoactive substances. Moreover, a set of normative instruments and multilateral bodies and systems were developed to help States implement and adjudicate such regulation. As a result, the three main drug control conventions, which form the foundation of the international drug control system, today enjoy near universal adherence, with more than 180 States parties. This volume presents an outline of the historical development of the modern drug control system: why and how it arose, its impact on drug production and consumption and its legacy for present and future international drug control efforts.

An evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity.

Foamy viruses (FVs) are highly fusogenic, and their replication induces massive syncytium formation in infected cell cultures which is believed to be mediated by expression of the envelope (Env) protein. The FV Env is essential for virus particle egress. The unusually long putative membrane-spanning domain (MSD) of the transmembrane subunit carries dispersed charged amino acids and has an important function for particle envelopment. To better understand the capsid-envelope interaction and Env-mediated cell fusion, we generated a variety of FV MSD mutations. C-terminal deletions revealed the cytoplasmic domain to be dispensable but the full-length MSD to be required for fusogenic activity. The N-terminal 15 amino acids of the MSD were found to be sufficient for membrane anchorage and promotion of FV particle release. Expression of wild-type Env protein rarely induced syncytia due to intracellular retention. Coexpression with FV Gag-Pol resulted in particle export and a dramatic increase in fusion activity. A nonconservative mutation of K(959) in the middle of the putative MSD resulted in increased fusogenic activity of Env in the absence of Gag-Pol due to enhanced cell surface expression as well as structural changes in the mutant proteins. Coexpression with Gag-Pol resulted in a further increase in the fusion activity of mutant FV Env proteins. Our results suggest that an interaction between the viral capsid and Env is required for FV-induced giant-cell formation and that the positive charge in the MSD is an important determinant controlling intracellular transport and fusogenic activity of the FV Env protein.

Foamy virus capsids require the cognate envelope protein for particle export.

Unlike other subclasses of the Retroviridae the Spumavirinae, its prototype member being the so-called human foamy virus (HFV), require the expression of the envelope (Env) glycoprotein for viral particle egress. Both the murine leukemia virus (MuLV) Env and the vesicular stomatitis virus G protein, which efficiently pseudotype other retrovirus capsids, were not able to support export of HFV particles. Analysis of deletion and point mutants of the HFV Env protein revealed that the HFV Env cytoplasmic domain (CyD) is dispensable for HFV particle envelopment, release, and infectivity, whereas deletion of the membrane-spanning-domain (MSD) led to an accumulation of naked capsids in the cytoplasm. Neither alternative membrane association of HFV Env deletion mutants lacking the MSD and CyD via phosphoglycolipid anchor nor domain swapping mutants, with the MSD or CyD of MuLV Env and VSV-G exchanged against the corresponding HFV domains, could restore particle envelopment and the release defect of pseudotypes. However, replacement of the HFV MSD with that of MuLV led to budding of HFV capsids at the intracellular membranes. These virions were of apparently wild-type morphology but were not naturally released into the supernatant and they were noninfectious.

Characterization of cell lines carrying self-replicating hepatitis C virus RNAs.

Subgenomic selectable RNAs of the hepatitis C virus (HCV) have recently been shown to self-replicate to high levels in the human hepatoma cell line Huh-7 (V. Lohmann, F. Körner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113, 1999). Taking advantage of this cell culture system that allows analyses of the interplay between HCV replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 year. During this time, we observed no signs of cytopathogenicity such as reduction of growth rates or ultrastructural changes. High levels of HCV RNAs were preserved in cells passaged under continuous selection. When selective pressure was omitted replicon levels dropped, but depending on culture conditions the RNAs persisted for more than 10 months. A tight coupling of the amounts of HCV RNA and proteins to host cell growth was observed. Highest levels were found in exponentially growing cells, followed by a sharp decline in resting cells, suggesting that cellular factors required for RNA replication and/or translation vary in abundance and become limiting in resting cells. Studies of polyprotein processing revealed rapid cleavages at the NS3/4A and NS5A/B sites resulting in a rather stable NS4AB5A precursor that was processed slowly into individual products. Half-lives (t(1/2)s) of mature proteins ranged from 10 to 16 h, with the exception of the hyperphosphorylated form of NS5A, which was less stable (t(1/2), approximately 7 h). Results of immunoelectron microscopy revealed an association of the majority of viral proteins with membranes of the endoplasmic reticulum, suggesting that this is the site of RNA replication. In summary, replicon-bearing cells are a good model for viral persistence, and they allow the study of various aspects of the HCV life cycle.

Efficient intracellular retrotransposition of an exogenous primate retrovirus genome.

The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to shuttle to the nucleus and integrate into host cell genomic DNA. While a potential intracellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably transduced, harboring 1 to approximately 10 vector integrants. Generation of the integrants depended on expression of functional capsid, reverse transcriptase and integrase proteins, and did not involve an extracellular step. PCR analysis of the U3 region of the 5' long terminal repeat and determination of proviral integration sites showed that a reverse transcription step had taken place to generate the integrants. Co-expression of a mutated envelope allowing particle egress and avoiding extracellular infection resulted in a significantly increased rescue of cells harboring integrants, suggesting that accumulation of proviruses via intracellular retrotransposition represents an integral part of the FV replication strategy.

Specific binding of recombinant foamy virus envelope protein to host cells correlates with susceptibility to infection.

The interaction of simian foamy viruses (FVs) with their putative cellular receptor(s) was studied with two types of recombinant envelope protein (Env). Transient expression of full-length Env in BHK-21 cells induced syncytia formation. However, selected stable transfectants fused with naive cells but not with each other. A soluble fusion protein of the Env surface domain with the Fc fragment of a human IgG1 heavy chain (EnvSU-Ig) was produced in the baculovirus expression system, purified to homogeneity, and used for binding and competition analyses. EnvSU-Ig but not unrelated Ig fusion proteins bound to cells specifically. Neutralizing serum blocked binding of EnvSU-Ig and, vice versa, serum-mediated neutralization was abrogated by the chimeric protein. Concomitant reduction of EnvSU-Ig binding and FV susceptibility was seen in Env-expressing target cells. Although EnvSU-Ig did not inhibit FV infection, very likely due to its displacement by multivalent virus-cell interactions, this divalent ligand should help to characterize functionally and to identify the ubiquitous FV receptor.

A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.

Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.