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Title: Towards the development of potent and selective inhibitors of hepatitis A and E virus
Other Titles: De ontwikkeling van krachtige en selectieve inhibitoren van hepatitis A en E virus
Authors: Debing, Yannick
Issue Date: 1-Apr-2015
Abstract: 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.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory of Virology and Chemotherapy (Rega Institute)

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