Daniel Todt

In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome

Objective Hepatitis E virus (HEV) infection can take chronic courses in immunocompromised patients potentially leading to liver cirrhosis and liver failure. Ribavirin (RBV) is currently the only treatment option for many patients, but treatment failure can occur which has been associated with the appearance of a distinct HEV polymerase mutant (G1634R). Here, we performed a detailed analysis of HEV viral intrahost evolution during chronic hepatitis E infections. Design Illumina deep sequencing was performed for the detection of intrahost variation in the HEV genome of chronically infected patients. Novel polymerase mutants were investigated in vitro using state-of-the-art HEV cell culture models. Results Together, these data revealed that (1) viral diversity differed markedly between patients but did not show major intraindividual short-term variations in untreated patients with chronic hepatitis E, (2) RBV therapy was associated with an increase in viral heterogeneity which was reversible when treatment was stopped, (3) the G1634R mutant was detectable as a minor population prior to therapy in patients who subsequently failed to achieve a sustained virological response to RBV therapy and (4) in addition to G1634R further dominant variants in the polymerase region emerged, impacting HEV replication efficiency in vitro. Conclusions In summary, this first investigation of intrahost HEV population evolution indicates that RBV causes HEV mutagenesis in treated patients and that an emergence of distinct mutants within the viral population occurs during RBV therapy. We also suggest that next-generation sequencing could be useful to guide personalised antiviral strategies.

Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. Infection usually leads to acute hepatitis that can become fulminant, particularly among pregnant women and in patients with preexisting liver disease, or may evolve to a chronic state, especially in immunosuppressed individuals. HEV has been shown to produce a range of extra‐hepatic manifestations including aplastic anaemia, acute thyroiditis, glomerulonephritis as well as neurological disorders such as Guillain‐Barré syndrome, neuralgic amyotrophy and encephalitis. The pathogenesis of these neurological injuries remains largely unknown, and it is also uncertain whether or not HEV can directly infect neuronal cells. In this study, we investigated whether HEV is capable of completing the viral life cycle in human neuronal‐derived cell lines such as neuroepithelioma (SK‐N‐MC), desmoplastic cerebellar medulloblastoma (DAOY), glioblastoma multiforme (DBTRG), glioblastoma astrocytoma (U‐373 MG) and oligodendrocytic (M03.13) cells. Following transfection of these cells with HEV Gaussia luciferase reporter virus, all tested cell lines supported HEV RNA replication. Furthermore, extra‐ and intracellular viral capsid was detected by an HEV antigen ELISA as a marker for virus assembly and release. Permissiveness for HEV cell entry could be demonstrated for the oligodendrocytic cell line M03.13. In conclusion, these results indicate that HEV tropism is not restricted to the liver and HEV can potentially complete the full viral life cycle in neuronal‐derived tissues explaining neurologic disorders during HEV infection.

Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication

ABSTRACT Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I interferon (IFN) has been evaluated in a few infected transplant patients in vivo. In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication. Simultaneous determination of interferon-stimulated gene (ISG) expression levels for all IFN types demonstrated efficient downregulation by HEV. Furthermore, different IFN-α subtypes were also able to block viral replication in combination with ribavirin. The IFN-α subtypes 2a and 2b exerted the strongest antiviral activity against HEV. In conclusion, these data demonstrate for the first time moderate anti-HEV activities of types II and III IFNs and different IFN-α subtypes. As HEV employed a potent anti-interferon mechanism by restricting ISG expression, exogenous application of IFNs as immunotherapy should be carefully assessed.

Interferon-inducible cholesterol-25-hydroxylase restricts hepatitis C virus replication through blockage of membranous web formation.

Hepatitis C virus (HCV) is a positive‐strand RNA virus that primarily infects human hepatocytes. Infections with HCV constitute a global health problem, with 180 million people currently chronically infected. Recent studies have reported that cholesterol 25‐hydroxylase (CH25H) is expressed as an interferon‐stimulated gene and mediates antiviral activities against different enveloped viruses through the production of 25‐hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remain elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes. In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. Levels of hCH25H messenger RNA were significantly up‐regulated both in HCV‐positive liver biopsies and in HCV‐infected primary human hepatocytes. The expression of hCH25H in primary human hepatocytes was primarily and transiently induced by type I interferon. Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype‐independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication, which was observed using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web, the HCV replication factory, independent of RNA replication. Conclusion: Infection with HCV causes up‐regulation of interferon‐inducible CH25H in vivo, and its product, 25HC, restricts HCV primarily at the level of RNA replication by preventing formation of the viral replication factory. (Hepatology 2015;62:702–714)

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.

Hepatitis E Virus (HEV) ORF2 Antigen Levels Differentiate Between Acute and Chronic HEV Infection

BACKGROUND Hepatitis E virus (HEV) genotype 3 infections are frequent in Europe and North America, with acute and chronic courses described in the literature. HEV RNA detection by real-time polymerase chain reaction (PCR) is the gold standard for diagnosis. Recently, an anti-HEV antigen (Ag)-specific enzyme-linked immunosorbent assay (ELISA) directed against the HEV capsid became commercially available. The effectiveness of anti-HEV Ag-specific ELISA at detecting HEV genotype 3 infections remains undefined. METHODS The performance of anti-HEV Ag-ELISA was compared with that of real-time PCR, using sera from a cohort of acutely infected individuals, in addition to a cohort of chronically infected patients undergoing ribavirin therapy. Furthermore, virion properties were evaluated by density fractionation. RESULTS Anti-HEV Ag-specific ELISA was less sensitive than real-time PCR at detection of HEV infection. Anti-HEV Ag-specific ELISA revealed significantly higher HEV Ag in chronically infected individuals as compared to acutely infected patients, with high sensitivity and specificity to distinguish acute from chronic HEV infection. Of note, HEV Ag remained detectable for >100 days after HEV RNA clearance in ribavirin-treated patients with chronic HEV. Density gradients revealed the presence of membrane-associated virions in the sera, with a different distribution as compared to HEV RNA. CONCLUSIONS The anti-HEV Ag-specific ELISA is less sensitive than HEV RNA real-time PCR but represents a useful tool to discriminate chronic from acute infection.

Mechanisms of methods for hepatitis C virus inactivation.

ABSTRACT Virus inactivation by chemical disinfectants is an important instrument for infection control in medical settings, but the mechanisms involved are poorly understood. In this study, we systematically investigated the effects of several antiviral treatments on hepatitis C virus (HCV) particles as model for enveloped viruses. Studies were performed with authentic cell culture-derived viruses, and the influence of chemical disinfectants, heat, and UV treatment on HCV was analyzed by the determination of infectious particles in a limiting-dilution assay, by quantitative reverse transcription-PCR, by core enzyme-linked immunosorbent assay, and by proteolytic protection assay. All different inactivation methods resulted in a loss of HCV infectivity by targeting different parts of the virus particle. Alcohols such as ethanol and 2-propanol did not affect the viral RNA genome integrity but disrupted the viral envelope membrane in a capsid protection assay. Heat and UV treatment of HCV particles resulted in direct damage of the viral genome since transfection of viral particle-associated RNA into permissive cells did not initiate RNA replication. In addition, heat incubation at 80°C disrupted the HCV envelope, rendering the viral capsid susceptible to proteolytic digest. This study demonstrated the molecular processes of viral inactivation of an enveloped virus and should facilitate the development of effective disinfection strategies in infection control not only against HCV but also against other enveloped viruses.

Immune protection against reinfection with nonprimate hepacivirus

Significance Hepatitis C virus (HCV) displays a narrow species tropism severely hampering development of small animal models that are required for vaccine and pathogenesis studies in vivo. The recent discoveries of HCV-related hepaciviruses in diverse hosts offer new opportunities with respect to the development of an immunocompetent animal model for HCV research. Among the hepaciviruses, the equine nonprimate hepacivirus (NPHV) represents the closest homolog of HCV discovered to date. We defined key aspects of natural immunity to NPHV challenge in the cognate host and provide evidence for natural protection from NPHV infection. Further characterization of the immune signatures that confer protection against NPHV could provide important information that may facilitate the development of new prophylactic strategies including protective vaccines against HCV. Hepatitis C virus (HCV) displays a restricted host species tropism and only humans and chimpanzees are susceptible to infection. A robust immunocompetent animal model is still lacking, hampering mechanistic analysis of virus pathogenesis, immune control, and prophylactic vaccine development. The closest homolog of HCV is the equine nonprimate hepacivirus (NPHV), which shares similar features with HCV and thus represents an animal model to study hepacivirus infections in their natural hosts. We aimed to dissect equine immune responses after experimental NPHV infection and conducted challenge experiments to investigate immune protection against secondary NPHV infections. Horses were i.v. injected with NPHV containing plasma. Flow cytometric analysis was used to monitor immune cell frequencies and activation status. All infected horses became viremic after 1 or 2 wk and viremia could be detected in two horses for several weeks followed by a delayed seroconversion and viral clearance. Histopathological examinations of liver biopsies revealed mild, periportally accentuated infiltrations of lymphocytes, macrophages, and plasma cells with some horses displaying subclinical signs of hepatitis. Following viral challenge, an activation of equine immune responses was observed. Importantly, after a primary NPHV infection, horses were protected against rechallenge with the homologous as well as a distinct isolate with only minute amounts of circulating virus being detectable.

Assessment of cross-species transmission of hepatitis C virus-related non-primate hepacivirus in a population of humans at high risk of exposure.

The recent discovery of hepatitis C virus (HCV)-related viruses in different animal species has raised new speculations regarding the origin of HCV and the possibility of a zoonotic source responsible for the endemic HCV transmission. As a consequence, these new findings prompt questions regarding the potential for cross-species transmissions of hepaciviruses. The closest relatives to HCV discovered to date are the non-primate hepaciviruses (NPHVs), which have been described to infect horses. To evaluate the risk of a potential zoonotic transmission, we analysed NPHV RNA and antibodies in humans with occupational exposure to horses in comparison with a low-risk group. Both groups were negative for NPHV RNA, even though low seroreactivities against various NPHV antigens could be detected irrespective of the group. In conclusion, we did not observe evidence of NPHV transmission between horses and humans.

Mutagenic Effects of Ribavirin on Hepatitis E Virus-Viral Extinction versus Selection of Fitness-Enhancing Mutations.

Hepatitis E virus (HEV), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. To date, HEV infections can only be treated with ribavirin (RBV). Major drawbacks of this therapy are that RBV is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to RBV. One of the proposed modes of action of RBV is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. These transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. In contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. Emergence of these mutant viruses can lead to therapeutic failure. Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis.