Yijin Wang

Ribavirin Inhibits In Vitro Hepatitis E Virus Replication through Depletion of Cellular GTP Pools and Is Moderately Synergistic with Alpha Interferon

ABSTRACT Hepatitis E virus (HEV) is a common cause of acute hepatitis that results in high mortality in pregnant women and may establish chronic infections in immunocompromised patients. We demonstrate for the first time that alpha interferon (IFN-α) and ribavirin inhibit in vitro HEV replication in both a subgenomic replicon and an infectious culture system based on a genotype 3 strain. IFN-α showed a moderate but significant synergism with ribavirin. These findings corroborate the reported clinical effectiveness of both drugs. In addition, the antiviral activity of ribavirin against wild-type genotype 1, 2, and 3 strains was confirmed by immunofluorescence staining. Furthermore, the in vitro activity of ribavirin depends on depletion of intracellular GTP pools, which is evident from the facts that (i) other GTP-depleting agents (5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide [EICAR] and mycophenolic acid) inhibit viral replication, (ii) exogenously added guanosine reverses the antiviral effects, and (iii) a strong correlation (R2 = 0.9998) exists between the antiviral activity and GTP depletion of ribavirin and other GTP-depleting agents.

Rapamycin and everolimus facilitate hepatitis e virus replication: Revealing a basal defense mechanism of PI3K-PKB-mTOR pathway

BACKGROUND & AIMS Humans are frequently exposed to hepatitis E virus (HEV). Nevertheless, the disease mainly affects pregnant women and immunocompromised individuals. Organ recipients receiving immunosuppressants, such as rapalogs, to prevent rejection have a high risk for developing chronic hepatitis following HEV infection. Rapalogs constitute potent inhibitors of mTOR including rapamycin and everolimus. As a master kinase, the mechanism-of-action of mTOR is not only associated with the immunosuppressive capacity of rapalogs but is also tightly regulated during pregnancy because of increased nutritional demands. METHODS We thus investigated the role of mTOR in HEV infection by using two state-of-the-art cell culture models: a subgenomic HEV containing luciferase reporter and a full-length HEV infectious cell culture system. RESULTS In both subgenomic and full-length HEV models, HEV infection was aggressively escalated by treatment of rapamycin or everolimus. Inhibition of mTOR was confirmed by Western blot showing the inhibition of its downstream target, S6 phosphorylation. Consistently, stable silencing of mTOR by lentiviral RNAi resulted in a significant increase in intracellular HEV RNA, suggesting an antiviral function of mTOR in HEV infection. By targeting a series of other up- and downstream elements of mTOR signaling, we further revealed an effective basal defense mechanism of the PI3K-PKB-mTOR pathway against HEV, which is through the phosphorylated eIF4E-binding protein 1 (4E-BP1), however independent of autophagy formation. CONCLUSIONS The discovery that PI3K-PKB-mTOR pathway limits HEV infection through 4E-BP1 and acts as a gate-keeper in human HEV target cells bears significant implications in managing immunosuppression in HEV-infected organ transplantation recipients.

Modeling rotavirus infection and antiviral therapy using primary intestinal organoids.

Despite the introduction of oral vaccines, rotavirus still kills over 450,000 children under five years of age annually. The absence of specific treatment prompts research aiming at further understanding of pathogenesis and the development of effective antiviral therapy, which in turn requires advanced experimental models. Given the intrinsic limitations of the classical rotavirus models using immortalized cell lines infected with laboratory-adapted strains in two dimensional cultures, our study aimed to model infection and antiviral therapy of both experimental and patient-derived rotavirus strains using three dimensional cultures of primary intestinal organoids. Intestinal epithelial organoids were successfully cultured from mouse or human gut tissues. These organoids recapitulate essential features of the in vivo tissue architecture, and are susceptible to rotavirus. Human organoids are more permissive to rotavirus infection, displaying an over 10,000-fold increase in genomic RNA following 24h of viral replication. Furthermore, infected organoids are capable of producing infectious rotavirus particles. Treatment of interferon-alpha or ribavirin inhibited viral replication in organoids of both species. Importantly, human organoids efficiently support the infection of patient-derived rotavirus strains and can be potentially harnessed for personalized evaluation of the efficacy of antiviral medications. Therefore, organoids provide a robust model system for studying rotavirus-host interactions and assessing antiviral medications.

CeO2 nanocubes-graphene oxide as durable and highly active catalyst support for proton exchange membrane fuel cell

Rapid degradation of cell performance still remains a significant challenge for proton exchange membrane fuel cell (PEMFC). In this work, we develop novel CeO2 nanocubes-graphene oxide nanocomposites as durable and highly active catalyst support for proton exchange membrane fuel cell. We show that the use of CeO2 as the radical scavenger in the catalysts remarkably improves the durability of the catalyst. The catalytic activity retention of Pt-graphene oxide-8 wt.% CeO2 nanocomposites reaches as high as 69% after 5000 CV-cycles at a high voltage range of 0.8–1.23 V, in contrast to 19% for that of the Pt-graphene oxide composites. The excellent durability of the Pt-CeO2 nanocubes-graphene oxide catalyst is attributed to the free radical scavenging activity of CeO2, which significantly slows down the chemical degradation of Nafion binder in catalytic layers, and then alleviates the decay of Pt catalysts, resulting in the excellent cycle life of Pt-CeO2-graphene oxide nanocomposite catalysts. Additionally, the performance of single cell assembled with Nafion 211 membrane and Pt-CeO2-graphene oxide catalysts with different CeO2 contents in the cathode as well as the Pt-C catalysts in the anode are also recorded and discussed in this study.

Emerging methanol-tolerant AlN nanowire oxygen reduction electrocatalyst for alkaline direct methanol fuel cell

Replacing precious and nondurable Pt catalysts with cheap materials is a key issue for commercialization of fuel cells. In the case of oxygen reduction reaction (ORR) catalysts for direct methanol fuel cell (DMFC), the methanol tolerance is also an important concern. Here, we develop AlN nanowires with diameters of about 100–150 nm and the length up to 1 mm through crystal growth method. We find it is electrochemically stable in methanol-contained alkaline electrolyte. This novel material exhibits pronounced electrocatalytic activity with exchange current density of about 6.52 × 10−8 A/cm2. The single cell assembled with AlN nanowire cathodic electrode achieves a power density of 18.9 mW cm−2. After being maintained at 100 mA cm−2 for 48 h, the AlN nanowire-based single cell keeps 92.1% of the initial performance, which is in comparison with 54.5% for that assembled with Pt/C cathode. This discovery reveals a new type of metal nitride ORR catalyst that can be cheaply produced from crystal growth method.

Hepatitis E virus infects neurons and brains.

Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.

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. FASEB J. 30, 3352–3367 (2016). www.fasebj.org

Convergent Transcription of Interferon-stimulated Genes by TNF-α and IFN-α Augments Antiviral Activity against HCV and HEV

IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for some cases of hepatitis E virus (HEV) infection. TNF-α is another important cytokine involved in inflammatory disease, which can interact with interferon signaling. Because interferon-stimulated genes (ISGs) are the ultimate antiviral effectors of the interferon signaling, this study aimed to understand the regulation of ISG transcription and the antiviral activity by IFN-α and TNF-α. In this study, treatment of TNF-α inhibited replication of HCV by 71 ± 2.4% and HEV by 41 ± 4.9%. Interestingly, TNF-α induced the expression of a panel of antiviral ISGs (2-11 fold). Blocking the TNF-α signaling by Humira abrogated ISG induction and its antiviral activity. Chip-seq data analysis and mutagenesis assay further revealed that the NF-κB protein complex, a key downstream element of TNF-α signaling, directly binds to the ISRE motif in the ISG promoters and thereby drives their transcription. This process is independent of interferons and JAK-STAT cascade. Importantly, when combined with IFN-α, TNF-α works cooperatively on ISG induction, explaining their additive antiviral effects. Thus, our study reveals a novel mechanism of convergent transcription of ISGs by TNF-α and IFN-α, which augments their antiviral activity against HCV and HEV.

Chronic hepatitis e in solid-organ transplantation: The key implications of immunosuppressants

Purpose of review Solid-organ recipients infected with hepatitis E virus (HEV) bear an extremely high risk of developing chronic hepatitis, although this virus only causes acute infection in the general population. Immunosuppressive medication universally used after transplantation to prevent organ rejection appears to be a main risk factor for developing chronic infection. This review aims to overview and emphasize the current clinical and experimental evidence regarding the key implications of immunosuppressants in chronic hepatitis E. Recent findings Over 60% of organ recipients who are infected with HEV develop chronic hepatitis. Immunosuppressant treatment after transplantation was identified as a key risk factor. Therefore, dose reduction or even withdrawal of immunosuppressants is considered as the first intervention strategy to achieve viral clearance in these patients. Otherwise, ribavirin, as an off-label medication, is considered as an antiviral treatment, with compelling outcomes observed so far. Interestingly, in addition to a common immunosuppression property that can favour HEV infection in general, different types of immunosuppressants may exert differential impacts on the infection course in patients. Furthermore, potential interaction may exist between particular immunosuppressant and ribavirin. With the recent development of a cell culture system for HEV, experimental research has been initiated to investigate how immunosuppressive drugs interact with HEV infection. Summary On the basis of the current evidence, it remains impossible to define an optimal immunosuppressive protocol for these HEV-infected patients. However, the realization of this clinical issue and the initiation of translational research using cell culture models of HEV have been represented as milestones in this field.

RIG‐I is a key antiviral interferon‐stimulated gene against hepatitis E virus regardless of interferon production

Interferons (IFNs) are broad antiviral cytokines that exert their function by inducing the transcription of hundreds of IFN‐stimulated genes (ISGs). However, little is known about the antiviral potential of these cellular effectors on hepatitis E virus (HEV) infection, the leading cause of acute hepatitis globally. In this study, we profiled the antiviral potential of a panel of important human ISGs on HEV replication in cell culture models by overexpression of an individual ISG. The mechanism of action of the key anti‐HEV ISG was further studied. We identified retinoic acid–inducible gene I (RIG‐I), melanoma differentiation–associated protein 5, and IFN regulatory factor 1 (IRF1) as the key anti‐HEV ISGs. We found that basal expression of RIG‐I restricts HEV infection. Pharmacological activation of the RIG‐I pathway by its natural ligand 5′‐triphosphate RNA potently inhibits HEV replication. Overexpression of RIG‐I activates the transcription of a wide range of ISGs. RIG‐I also mediates but does not overlap with IFN‐α‐initiated ISG transcription. Although it is classically recognized that RIG‐I exerts antiviral activity through the induction of IFN production by IRF3 and IRF7, we reveal an IFN‐independent antiviral mechanism of RIG‐I in combating HEV infection. We found that activation of RIG‐I stimulates an antiviral response independent of IRF3 and IRF7 and regardless of IFN production. However, it is partially through activation of the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) cascade of IFN signaling. RIG‐I activated two distinct categories of ISGs, one JAK‐STAT‐dependent and the other JAK‐STAT‐independent, which coordinately contribute to the anti‐HEV activity. Conclusion: We identified RIG‐I as an important anti‐HEV ISG that can be pharmacologically activated; activation of RIG‐I stimulates the cellular innate immunity against HEV regardless of IFN production but partially through the JAK‐STAT cascade of IFN signaling. (Hepatology 2017;65:1823‐1839).