Towards the development of potent and selective inhibitors of hepatitis A and E virus

Hepatitis A and E virus are the most prominent¨ causes of acute viral hepatitis worldwide (chapter ¨1). Despite the availability of an effective vacc ine for hepatitis A virus (HAV), it is widespread¨ in developing countries and outbreaks still occur¨ regularly in industrialized regions. Although most ¨infections are asymptomatic, or have a mild clini cal course, severe cases of hepatitis A may occur, ¨especially in the elderly. The availability of po tent and selective inhibitors of HAV replication w ould be highly appreciated for treating these seve re cases, to reduce the duration of illness and to ¨contain outbreaks. To allow for identi fication and development of such inhibitors of HAV ¨replication, three antiviral assays were develope d, including a cytopathic effect (CPE) reduction a ssay and two virus yield reduction assays for geno types IB and IIIA (chapter 2). The assays were val idated with two known inhibitors of HAV replicatio n: interferon alpha (IFNα) effectively inhib ited virus replication, while the activity of aman tadine HCl proved to be strain-dependent. In addit ion, the effect of the enterovirus inhibitors plec onaril, rupintrivir and enviroxime on HAV replicat ion was assessed. Pleconaril exhibited some very m oderate activity, whereas the effect of rupintrivi r proved to be strain-dependent. Enviroxime did no t inhibit HAVreplication, suggesting that phosphat idylinositol-4-kinase IIIβ (PI4KIIIβ) is¨not crucial in the HAV life cycle. Sim ilar to HAV, hepatitis E virus (HEV) is transmitte d feco-orally. Consequently, large outbreaks are o ften reported in refugee camps, e.g. in Africa. In fections are often asymptomatic or mild, although¨ severe infections may occur, especially in pregnan t women. Chronic cases of hepatitis E have also be en described in immunocompromised patients (such a s transplant recipients) with a high risk for cirr hosis and liver failure. HEV infections can be tre ated with an extended ribavirin (RBV) regimen whic h seems mostlyeffective, although cases of treatme nt failure have been reported and side effects may ¨be severe. Consequently, there is a need for more¨potentnon-toxic inhibitors of HEV replication and ¨for characterization of thecurrently used RBV tre atment regimen. First, an antiviral ass ay was developed using the HEV-related cutthroat t rout virus (CTV) as a surrogate virus (chapter 3). ¨RBV and trout IFN were found to efficiently inhib it CTV replication, while other known broad-spectr um inhibitorsof RNA virus replication such as the¨ nucleoside analog 2’-C-methylcytidine result ed only in a moderate antiviral activity. In addit ion, CTV is only detected in trout during spawning , which suggests a hormonal influence on viral rep lication and is reminiscent of the high susceptibi lity of pregnant women to severe hepatitis E. Cons equently, the effect of three sex steroids on in vitro CTV replication was evaluated. Where as progesterone resulted in marked inhibition of v irus replication, testosterone and 17β-estrad iol stimulated viral growth. Our data thusindicate ¨that CTV may serve as a surrogate model for HEV. Next, the in vitro culturable¨ HEV strain Kernow-C1 p6 and a derived subgenomic r eplicon were used to develop antiviral assays for¨ the genuine HEV (chapter 4). Thus it was confirmed ¨that IFNα and RBV inhibit in vitro ¨HEV replication. A moderate but significant syner gism was observed for combinations of both drugs.¨ These findings corroborate the reported clinical e ffectiveness. In addition, the invitro me chanism of action of RBV was studied and was found¨to dependon depletion of intracellular GTP pools. Since cases of RBV treatment failure h ave been described, a collaboration was initiated¨ with the clinical reference laboratory for hepatit is E in Hannover, Germany (chapter 5). We analyzed ¨blood samples collected from 15 patients with chr onic hepatitis E who were recipients of solid-orga n transplants andwere treated with RBV. All patien ts cleared the virus except for two non-responders ¨of which one patient died. A G1634R mutation in t he viral polymerase was detected in the HEV RNA of ¨the non-responders; this mutation did not provide ¨the virus with resistance to RBV in vitro.However, the mutant form of a subgenomic replico n of genotype 3 HEV replicated more efficiently in vitro than HEV without this mutation, an d the same was true for infectious virus, includin g in competition assays. Similar results were obta ined for genotype 1 HEV. The G1634R mutation there fore appears to increase the replicative capacity¨ of HEV inthe human liver and hence reduce the effi cacy of RBV. In a recent paper by Zhou¨ and colleagues, a stimulating effect of mammalian¨ targetof rapamycin (mTOR)-inhibitors such as rapam ycin and everolimus on HEV replication was describ ed. In chapter 6, we discuss these observatio ns, including clinical implications and possible u nderlying mechanisms, in an Editorial. Finally, chapter 7 comprises the announcement ¨of the first complete genome sequence of a US iso late ofrat HEV. This is a first step towards the i mplementation of a convenient small animal model f or hepatitis E.status: publishe

Antiviral strategies for hepatitis E virus

The hepatitis E virus is a common cause of acute hepatitis. Contrary to hepatitis B and C, hepatitis E is mostly a mild infection, although it has a high mortality in pregnant women and can evolve to chronicity in immunocompromised patients. Ribavirin and pegylated interferon-α are the only available therapies, but both have side effects that are not acceptable for prophylaxis or treatment of mild infections. In addition, these drugs cannot be used for all patient types (e.g. in case of pregnancy, specific organ transplants or co-morbidities) and in resource-poor settings. Hence there is an urgent need for better antiviral treatments that are efficacious and safe, also during pregnancy. In this review, a concise introduction to the virus and disease is provided, followed by a discussion of the available assay systems and potential molecular targets (viral proteins and host factors) for the development of inhibitors of HEV replication. Finally, directions for future research are presented.publisher: Elsevier articletitle: Antiviral strategies for hepatitis E virus journaltitle: Antiviral Research articlelink: http://dx.doi.org/10.1016/j.antiviral.2013.12.005 content_type: article copyright: Copyright © 2014 Elsevier B.V. All rights reserved.status: publishe

mTOR-inhibitors may aggravate chronic hepatitis E

publisher: Elsevier articletitle: mTOR-inhibitors may aggravate chronic hepatitis E journaltitle: Journal of Hepatology articlelink: http://dx.doi.org/10.1016/j.jhep.2014.07.016 associatedlink: http://dx.doi.org/10.1016/j.jhep.2014.05.026 content_type: simple-article copyright: © 2014 European Association for the Study of the Liver. Published by Elsevier B.V.status: publishe

Intervention strategies for emerging viruses: use of antivirals

Today, small molecule antiviral drugs are available for the treatment of infections with herpesviruses, HIV, HBV and HCV as well as with influenza viruses. Ribavirin, a broad-spectrum (but aspecific) antiviral, has been approved for the treatment of infections with respiratory syncytial virus, HCV and Lassa virus. Yet, for many other viruses that cause life-threatening infections [most of which are considered emerging and/or neglected] there are no drugs available. Ideally, potent and broad-spectrum (i.e., pan-genus or pan-family virus activity) antiviral drugs should be developed whereby one drug could be used for the treatment of a number of such viral infections. We here review recent evolutions in the search for inhibitors of emerging and neglected RNA viruses.status: publishe

Molecular biology and inhibitors of hepatitis A virus

Hepatitis A virus (HAV) is a faeco-orally transmitted picornavirus and is one of the main causes of acute hepatitis worldwide. An overview of the molecular biology of HAV is presented with an emphasis on recent findings. Immune evasion strategies and a possible correlation between HAV and atopy are discussed as well. Despite the availability of efficient vaccines, antiviral drugs targeting HAV are required to treat severe cases of fulminant hepatitis, contain outbreaks, and halt the potential spread of vaccine-escape variants. Additionally, such drugs could be used to shorten the period of illness and decrease associated economical costs. Several known inhibitors of HAV with various mechanisms of action will be discussed. Since none of these molecules is readily useable in the clinic and since the availability of an anti-HAV drug would be of clinical importance, increased efforts should be targeted toward discovery and development of such antivirals.status: publishe

New models to study hepatitis E virus replication and particular characteristics of infection: The beedle hides in the hay stack

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Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2'-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.Link to journal homepage: http://www.journals.elsevier.com/antiviral-research/status: publishe

Rapid and convenient assays to assess potential inhibitory activity on in vitro hepatitis A replication

Three different antiviral assays were developed for the in vitro screening of inhibitors of the hepatitis A virus (HAV) of which (i) a cytopathic effect reduction assay suitable for medium-to-high-throughput screening and (ii) two virus yield reduction assays (based on quantification of viral RNA) for genotypes IB and IIIA. The assays were validated for antiviral studies with interferon-alpha (IFNα) and amantadine HCl, two known inhibitors of HAV replication. IFNα effectively inhibited HAV replication, whereas the activity of amantadine HCl appeared to be strain-dependent. Employing these assays, we assessed the effect of the known enterovirus inhibitors pleconaril, rupintrivir and enviroxime on HAV replication. Pleconaril exhibited some very moderate activity, the effect of rupintrivir proved to be strain-dependent. Enviroxime did not inhibit HAV replication, suggesting that phosphatidylinositol-4-kinase IIIβ is not crucial in the HAV life cycle.Link to journal homepage: http://www.journals.elsevier.com/antiviral-research/status: publishe

A rat model for hepatitis E virus

Hepatitis E virus (HEV) is one of the prime causes of acute viral hepatitis, whereas chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone pLA-B350 was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo Furthermore, a subgenomic replicon pLA-B350/luc was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of alpha interferon, ribavirin and mycophenolic acid, than genotype 3 HEV. As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 is amenable to humanization. Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies.status: publishe