Inhibits Hepatitis E Virus Replication Research Articles

High dose sofosbuvir and sofosbuvir-plus-ribavirin therapy inhibit Hepatitis E Virus (HEV) replication in a rabbit model for acute HEV infection

Hepatitis E virus (HEV) is an important cause of viral hepatitis worldwide and there is currently no FDA-approved anti-HEV drug. The commonly used drug ribavirin (RBV) could not achieve viral clearance in all patients and can induce drug resistance. Recent studies showed sofosbuvir (SOF) can inhibit HEV replication in vitro and has add-on effect when combined with RBV, but the clinical effect of SOF against HEV infection remains controversial and the dosage of SOF warrants further exploration. In this study, a rabbit model for acute HEV infection was used to evaluate the effect of SOF at different doses against HEV genotype 3 and 4, and to compare the antiviral effect of SOF-plus-RBV therapy with RBV monotherapy. Virological parameters on fecal, serological and intrahepatic level were tested by real-time PCR and ELISA. Liver function tests and histopathological assays were performed. Both 200 mg/d and 300 mg/d SOF treatment inhibits HEV replication with relieved liver inflammation and declined levels of fecal HEV RNA and antigenemia. 300 mg/d SOF eliminated HEV replication while a short viral rebound was observed after 200 mg/d SOF treatment. The SOF-plus-RBV therapy also showed stronger anti-HEV effect than RBV monotherapy. Our study suggests that high dose of SOF showed anti-HEV effect in the rabbit model. Moreover, the de novo SOF-plus-RBV therapy which eliminated acute HEV infection more efficiently than RBV monotherapy may serve as an alternative treatment strategy but warrants further preclinical and clinical study.

Niclosamide inhibits hepatitis E virus through suppression of NF-kappaB signalling

Hepatitis E virus (HEV) infection can cause severe acute hepatitis in pregnant women and chronic infection in immunocompromised patients, promoting the development of effective antiviral therapies. In this study, we identified niclosamide, a widely used anthelmintic drug, as a potent inhibitor of HEV replication in a range of subgenomic and full-length HEV models, which are based on human cell lines and liver organoids harbouring genotype 1 and 3 HEV strains. Niclosamide is known to have multiple cellular targets including the inhibition of STAT3 and NFκB signaling pathways. Although HEV activates STAT3, we excluded its involvement in the anti-HEV activity of niclosamide. Interestingly, HEV infection activated NFκB and activation of NFκB promoted viral replication. Consistently, stable silencing of NFκB by lentiviral RNAi inhibited HEV replication. By targeting NFκB signaling, we further revealed its role in mediating the anti-HEV action of niclosamide. These results demonstrated that niclosamide potently inhibits HEV replication by inhibiting NFκB signaling but independent of STAT3. Our findings support the potential of repurposing niclosamide for treating HEV infection.

The macrolide antibiotic azithromycin potently inhibits hepatitis E virus in cell culture models

Hepatitis E virus (HEV) infection in immunocompromised patients, pregnant women and children requires treatment; however, no approved medication is currently available. The macrolide antibiotic azithromycin has been identified as a potent HEV inhibitor. Azithromycin inhibits HEV replication and viral protein expression in multiple cell culture models with genotype 1 and 3 strains. This is largely independent of its induction of an interferon-like response. Because it is safe and cheap, repurposing azithromycin for treating HEV infection is attractive, particularly in resource-limited settings.

Inhibition of hepatitis E virus replication by zinc-finger antiviral Protein synergizes with IFN-β.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. However, host-HEV interactions have yet to be fully understood. Zinc-finger antiviral protein (ZAP) is a novel interferon (IFN)-stimulated gene product that inhibits a variety of viruses in synergy with IFN-β. To evaluate the role of ZAP in HEV infection, its expressions in HEV-infected patients and in cell cultures were measured. We report a significant inhibition of ZAP expression in patients with HEV genotype four acute infection. The expression of ZAP in the HEV life cycle was monitored in cultures of HEV-infected cells. Results indicated that the ZAP level decreased significantly after HEV infection. ZAP over-expression inhibited HEV replication, whereas its knockdown by RNA interference significantly increased HEV RNA. These suggest that ZAP serves as an antiviral in HEV infection. Moreover, silencing ZAP decreased IFN regulatory factor 3 (IRF3) phosphorylation in HEV-infected cells treated with poly(I:C), indicating that ZAP synergizes with IFN-β. In conclusion, ZAP is an important anti-HEV host factor and in synergy with IFN-β, inhibits HEV replication.

Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90

Hepatitis E virus (HEV) causes 14 million infections and 60,000 deaths per year globally, with immunocompromised persons and pregnant women experiencing severe symptoms. Although ribavirin can be used to treat chronic hepatitis E, toxicity in pregnant patients and the emergence of resistant strains are major concerns. Therefore there is an imminent need for effective HEV antiviral agents. The aims of this study were to develop a drug screening platform and to discover novel approaches to targeting steps within the viral life cycle. We developed a screening platform for molecules inhibiting HEV replication and selected a candidate, isocotoin. Isocotoin inhibits HEV replication through interference with heat shock protein 90 (HSP90), a host factor not previously known to be involved in HEV replication. Additional work is required to understand the compound's translational potential, however this suggests that HSP90-modulating molecules, which are in clinical development as anti-cancer agents, may be promising therapies against HEV.

Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation

Exposure to hepatitis E virus (HEV) bears a high risk of developing chronic infection in immunocompromised patients, including organ transplant recipients and cancer patients. We aim to identify effective anti-HEV therapies through screening and repurposing safe-in-human broad-spectrum antiviral agents. In this study, a safe-in-human broad-spectrum antiviral drug library comprising of 94 agents was used. Upon screening, we identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of HEV replication. The antiviral effect was confirmed in a range of cell culture models with genotype 1 and 3 HEV strains. As a cytidine analog, exogenous supplementation of pyrimidine nucleosides effectively reversed the antiviral activity of gemcitabine, but the level of pyrimidine nucleosides per se does not affect HEV replication. Surprisingly, similar to interferon-alpha (IFNα) treatment, gemcitabine activates STAT1 phosphorylation. This subsequently triggers activation of interferon-sensitive response element (ISRE) and transcription of interferon-stimulated genes (ISGs). Cytidine or uridine effectively inhibits gemcitabine-induced activation of ISRE and ISGs. As expected, JAK inhibitor 1 blocked IFNα, but not gemcitabine-induced STAT1 phosphorylation, ISRE/ISG activation, and anti-HEV activity. These effects of gemcitabine were completely lost in STAT1 knockout cells. In summary, gemcitabine potently inhibits HEV replication by triggering interferon-like response through STAT1 phosphorylation but independent of Janus kinases. This represents a non-canonical antiviral mechanism, which utilizes the innate defense machinery that is distinct from the classical interferon response. These results support repurposing gemcitabine for treating hepatitis E, especially for HEV-infected cancer patients, leading to dual anti-cancer and antiviral effects.

Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing.

The processing of polyprotein(s) to form structural and non-structural components remains an enigma due to the non-existence of an efficient and robust Hepatitis E Virus (HEV) culture system. We used the BacMam approach to construct an HEV replication model in which the HEV genome was cloned in the BacMam vector under the CMV promoter. The recombinant BacMam was used to infect Huh7 cells to transfer the HEV genome. HEV replication was authenticated by the presence of RNAs of both the polarity (+) and (−) and formation of hybrid RNA, a replication intermediate. The presence of genes for Papain-like Cysteine Protease (PCP), methyltransferase (MeT), RNA dependent RNA polymerase (RdRp), and ORF2 was confirmed by PCR amplification. Further, the infectious nature of the culture system was established as evidenced by the cross-infection of uninfected cells using the cell lysate from the infected cells. The HEV replication model was validated by detection of the ORF1 (Open Reading Frame1) encoded proteins, identified by Western blotting and Immunofluorescence by using epitope-specific antibodies against each protein. Consequently, discrete bands of 18, 35, 37, and 56 kDa corresponding to PCP, MeT, RdRp, and ORF2, respectively, were seen. Besides demonstrating the presence of non-structural enzymes of HEV along with ORF2, activity of a key enzyme, HEV-methyltransferase has also been observed. A 20% decrease in the replicative forms of RNA could be seen in presence of 100 μM Ribavirin after 48 h of treatment. The inhibition gradually increased from 0 to 24 to 48 h post-treatment. Summarily, infectious HEV culture system has been established, which could demonstrate the presence of HEV replicative RNA forms, the structural and non-structural proteins and the methyltransferase in its active form. The system may also be used to study the mechanism of action of Ribavirin in inhibiting HEV replication and develop a therapy.

Japonicone V, a sesquiterpene lactone derivative from the flowers of Inula japonica, inhibits hepatitis E virus replication by targeting virus-associated autophagy

Abstract Hepatitis E virus (HEV) is one of the major causes of acute hepatitis worldwide. Here, we investigated the role of autophagy on HEV replication and demonstrated anti-HEV effects of japonicone V, a main constituent of the flowers of Inula japonica. Results showed that HEV infection induced autophagy in Huh7 cells. Chloroquine (CQ) and 3-methyladenine (3-MA), two autophagy inhibitors inhibited HEV replication. Moreover, japonicone V suppressed HEV replication and inhibited autophagy in the HEV infectious model. CQ had additive inhibitory effects with japonicone V on HEV replication, while rapamycin, an autophagy inducer, attenuated japonicone V-mediated inhibition of HEV replication. In addition, HEV infection inhibited the PI3K/AKT/mTOR pathway; however, this effect could be alleviated by japonicone V treatment. Collectively, our findings reveal that autophagy machinery is required for HEV replication; japonicone V inhibited HEV replication by targeting the virus-associated autophagy, at least in part, through inhibition of the PI3K/AKT/mTOR pathway.

Zinc: A Potential Antiviral Against Hepatitis E Virus Infection?

Hepatitis E virus (HEV) is a major cause of viral hepatitis worldwide. Owing to its feco oral transmission route, sporadic as well as epidemic outbreaks recurrently occur. No specific antiviral therapy is available against the disease caused by HEV. Broad spectrum antivirals such as ribavirin and interferon alfa are prescribed in severe and chronic HEV cases. However, the side effects, cost, and limitations of usage render the available treatment unsuitable for several categories of patients. We recently reported the ability of zinc to inhibit viral replication in mammalian cell culture models of HEV infection. Zinc will be a safe and economical antiviral therapy option if it inhibits HEV replication during the natural course of infection. This essay discusses the putative mechanism(s) by which zinc inhibits HEV replication and provides an overview of the possible therapeutic potential of zinc in HEV patients.

Increased oestradiol in hepatitis E virus-infected pregnant women promotes viral replication.

Hepatitis E virus (HEV) infection causes subclinical diseases, leading to high mortality (>25%) in pregnant women. HEV replication is aggressively escalated in pregnant women, especially in the third trimester of pregnancy. Oestrogen plays an important role in pregnancy. However, the pathogenesis of HEV in pregnant women or immunosuppressive pregnant women (such as HIV-infected or organ-transplanted pregnant women) remains unclear. We investigated the role of oestradiol in HEV infection in a cell culture system. HEV-infected pregnant women had significantly higher oestradiol levels compared with uninfected individuals. HEV infection was significantly increased in cells treated with analogues of oestradiol, diethylstilbestrol (DES) or 17β-oestradiol in a dose-dependent way. However, tamoxifen, an antagonist oestrogen, inhibited HEV replication. HEV infection inhibits oestrogen receptor (ER-α) expression. Immunofluorescence and co-immunoprecipitation assays indicated that ER-α interacted with the helicase of HEV ORF1 indirectly. More importantly, HEV infection was exacerbated in immunosuppressive cells treated with an inhibitor of PI3K-AKT-mTOR signal pathway (LY296004) and supplemented with pregnant women serum with high oestradiol simultaneously. These results strongly suggest that pregnant women with high oestradiol and/or immunosuppression will be vulnerable to HEV infection.

Identification of GBF1 as a cellular factor required for hepatitis E virus RNA replication.

The hepatitis E virus (HEV) genome is a single‐stranded, positive‐sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear, and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose‐dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wild type GBF1 or a BFA‐resistant GBF1 mutant rescuing HEV replication in BFA‐treated cells, confirmed that GBF1 is the only BFA‐sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalise with the ORF1 protein, and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1‐regulated mechanisms.

Biological or pharmacological activation of protein kinase C alpha constrains hepatitis E virus replication

Although hepatitis E has emerged as a global health issue, there is limited knowledge of its infection biology and no FDA-approved medication is available. Aiming to investigate the role of protein kinases in hepatitis E virus (HEV) infection and to identify potential antiviral targets, we screened a library of pharmacological kinase inhibitors in a cell culture model, a subgenomic HEV replicon containing luciferase reporter. We identified protein kinase C alpha (PKCα) as an essential cell host factor restricting HEV replication. Both specific inhibitor and shRNA-mediated knockdown of PKCα enhanced HEV replication. Conversely, over-expression of the activated form of PKCα or treatment with its pharmacological activator strongly inhibited HEV replication. Interestingly, upon the stimulation by its activator, PKCα efficiently activates its downstream Activator Protein 1 (AP-1) pathway, leading to the induction of antiviral interferon-stimulated genes (ISGs). This process is independent of the JAK-STAT machinery and interferon production. However, PKCα induced HEV inhibition appears independent of the AP1 cascade. The discovery that activated PKCα restricts HEV replication reveals new insight of HEV-host interactions and provides new target for antiviral drug development.

IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

Disparity of basal and therapeutically activated interferon signalling in constraining hepatitis E virus infection.

Hepatitis E virus (HEV) represents one of the foremost causes of acute hepatitis globally. Although there is no proven medication for hepatitis E, pegylated interferon-α (IFN-α) has been used as off-label drug for treating HEV. However, the efficacy and molecular mechanisms of how IFN signalling interacts with HEV remain undefined. As IFN-α has been approved for treating chronic hepatitis C (HCV) for decades and the role of interferon signalling has been well studied in HCV infection, this study aimed to comprehensively investigate virus-host interactions in HEV infection with focusing on the IFN signalling, in comparison with HCV infection. A comprehensive screen of human cytokines and chemokines revealed that IFN-α was the sole humoral factor inhibiting HEV replication. IFN-α treatment exerted a rapid and potent antiviral activity against HCV, whereas it had moderate and delayed anti-HEV effects in vitro and in patients. Surprisingly, blocking the basal IFN pathway by inhibiting JAK1 to phosphorylate STAT1 has resulted in drastic facilitation of HEV, but not HCV infection. Gene silencing of the key components of JAK-STAT cascade of the IFN signalling, including JAK1, STAT1 and interferon regulatory factor 9 (IRF9), stimulated HEV infection. In conclusion, compared to HCV, HEV is less sensitive to IFN treatment. In contrast, the basal IFN cascade could effectively restrict HEV infection. This bears significant implications in management of HEV patients and future therapeutic development.

Calcineurin Inhibitors Stimulate and Mycophenolic Acid Inhibits Replication of Hepatitis E Virus

Many recipients of organ transplants develop chronic hepatitis, due to infection with the hepatitis E virus (HEV). Although chronic HEV infection is generally associated with immunosuppressive therapies, little is known about how different immunosuppressants affect HEV infection. A subgenomic HEV replication model, in which expression of a luciferase reporter gene is measured, and a full-length infection model were used. We studied the effects of different immunosuppressants, including steroids, calcineurin inhibitors (tacrolimus [FK506] and cyclosporin A), and mycophenolic acid (MPA, an inhibitor of inosine monophosphate dehydrogenase) on HEV replication in human hepatoma cell line Huh7. Expression of cyclophilins A and B (the targets of cyclosporin A) were knocked down using small hairpin RNAs. Steroids had no significant effect on HEV replication. Cyclosporin A promoted replication of HEV in the subgenomic and infectious models. Knockdown of cyclophilin A and B increased levels of HEV genomic RNA by 4.0- ± 0.6-fold and 7.2- ± 1.9-fold, respectively (n= 6; P < .05). A high dose of FK506 promoted infection of liver cells with HEV. In contrast, MPA inhibited HEV replication. Incubation of cells with guanosine blocked the antiviral activity of MPA, indicating that the antiviral effects of this drug involve nucleotide depletion. The combination of MPA and ribavirin had a greater ability to inhibit HEV replication than MPA or ribavirin alone. Cyclophilins A and B inhibit replication of HEV; this might explain the ability of cyclosporin A to promote HEV infection. On the other hand, the immunosuppressant MPA inhibits HEV replication. These findings should be considered when physicians select immunosuppressive therapies for recipients of organ transplants who are infected with HEV.

The Nucleotides on the Stem-Loop RNA Structure in the Junction Region of the Hepatitis E Virus Genome Are Critical for Virus Replication

The roles of conserved nucleotides on the stem-loop (SL) structure in the intergenic region of the hepatitis E virus (HEV) genome in virus replication were determined by using Huh7 cells transfected with HEV SL mutant replicons containing reporter genes. One or two nucleotide mutations of the AGA motif on the loop significantly reduced HEV replication, and three or more nucleotide mutations on the loop abolished HEV replication. Mutations on the stem and of the subgenome start sequence also significantly inhibited HEV replication. The results indicated that both the sequence and the SL structure in the junction region play important roles in HEV replication.