Yuebang Yin

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.

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.

Rotavirus in Organ Transplantation: Drug-Virus-Host Interactions

Although rotavirus is usually recognized as the most common etiology of diarrhea in young children, it can in fact cause severe diseases in organ transplantation recipients irrespective of pediatric or adult patients. This comprehensive literature analysis revealed 200 cases of rotavirus infection with 8 related deaths in the setting of organ transplantation been recorded. Based on published cohort studies, an average incidence of 3% (187 infections out of 6176 organ recipients) was estimated. Rotavirus infection often causes severe gastroenteritis complications and occasionally contributes to acute cellular rejection in these patients. Immunosuppressive agents, universally used after organ transplantation to prevent organ rejection, conceivably play an important role in such a severe pathogenesis. Interestingly, rotavirus can in turn affect the absorption and metabolism of particular immunosuppressive medications via several distinct mechanisms. Even though rotaviral enteritis is self‐limiting in general, infected transplantation patients are usually treated with intensive care, rehydration and replacement of nutrition, as well as applying preventive strategies. This article aims to properly assess the clinical impact of rotavirus infection in the setting of organ transplantation and to disseminate the interactions among the virus, host and immunosuppressive medications.

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).

Unphosphorylated ISGF3 drives constitutive expression of interferon-stimulated genes to protect against viral infections

A tripartite transcription factor complex mediates an interferon-independent antiviral response. An interferon-independent antiviral defense Virally infected cells produce type I interferons (IFNs), which stimulate the phosphorylation and activation of the STAT1 and STAT2 transcription factors. When combined with the transcriptional regulator IRF9, phosphorylated STAT1 and STAT2 form the ISGF3 complex, which drives the expression of IFN-stimulated genes (ISGs) that are important for antiviral immunity. Wang et al. report the formation of an alternative form of ISGF3, U-ISGF3, which drove the expression of ISGs and protected cells from viral infection in the absence of detection of detectable IFN or IFN signaling. In addition to IRF9, U-ISGF3 contained unphosphorylated STAT1 and STAT2. Together, these data suggest that U-ISGF3 drives constitutive expression of ISGs as part of an IFN-independent, antiviral immune response. Interferon (IFN)–stimulated genes (ISGs) are antiviral effectors that are induced by IFNs through the formation of a tripartite transcription factor ISGF3, which is composed of IRF9 and phosphorylated forms of STAT1 and STAT2. However, we found that IFN-independent ISG expression was detectable in immortalized cell lines, primary intestinal and liver organoids, and liver tissues. The constitutive expression of ISGs was mediated by the unphosphorylated ISGF3 (U-ISGF3) complex, consisting of IRF9 together with unphosphorylated STAT1 and STAT2. Under homeostatic conditions, STAT1, STAT2, and IRF9 were found in the nucleus. Analysis of a chromatin immunoprecipitation sequencing data set revealed that STAT1 specifically bound to the promoters of ISGs even in the absence of IFNs. Knockdown of STAT1, STAT2, or IRF9 by RNA interference led to the decreased expression of various ISGs in Huh7.5 human liver cells, which was confirmed in mouse embryonic fibroblasts (MEFs) from STAT1−/−, STAT2−/−, or IRF9−/− mice. Furthermore, decreased ISG expression was accompanied by increased replication of hepatitis C virus and hepatitis E virus. Conversely, simultaneous overexpression of all ISGF3 components, but not any single factor, induced the expression of ISGs and inhibited viral replication; however, no phosphorylated STAT1 and STAT2 were detected. A phosphorylation-deficient STAT1 mutant was comparable to the wild-type protein in mediating the IFN-independent expression of ISGs and antiviral activity, suggesting that ISGF3 works in a phosphorylation-independent manner. These data suggest that the U-ISGF3 complex is both necessary and sufficient for constitutive ISG expression and antiviral immunity under homeostatic conditions.

PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target

ABSTRACT Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, antiviral therapy is an important complement that cannot be substituted. Because of the lack of specific approved treatment, it is urgent to facilitate the cascade of further understanding of the infection biology, identification of druggable targets and the final development of effective antiviral therapies. PI3K-Akt-mTOR signaling pathway plays a vital role in regulating the infection course of many viruses. In this study, we have dissected the effects of PI3K-Akt-mTOR signaling pathway on rotavirus infection using both conventional cell culture models and a 3D model of human primary intestinal organoids. We found that PI3K-Akt-mTOR signaling is essential in sustaining rotavirus infection. Thus, blocking the key elements of this pathway, including PI3K, mTOR and 4E-BP1, has resulted in potent anti-rotavirus activity. Importantly, a clinically used mTOR inhibitor, rapamycin, potently inhibited both experimental and patient-derived rotavirus strains. This effect involves 4E-BP1 mediated induction of autophagy, which in turn exerts anti-rotavirus effects. These results revealed new insights on rotavirus-host interactions and provided new avenues for antiviral drug development.

Opposing Effects of Nitazoxanide on Murine and Human Norovirus

_To the Editor_ Norovirus is a major cause of acute gastroenteritis worldwide, and chronic infections frequently occur in immunocompromised patients. However, no US Food and Drug Administration–approved specific medication is available for treating norovirus infection. Interestingly, substantial clinical evidence has suggested that nitazoxanide, originally developed as an antiprotozoal agent, is a potential antiviral therapy for norovirus infection. Both clinical trials and case studies have reported its effects on reduction of symptom duration in immunocompetent patients or clearance of the infection in immunocompromised patients. On the contrary, a recent study in the Journal of Infectious Diseases by Kempf et al reported that nitazoxanide was not effective for treating chronic norovirus gastroenteritis in a patient with X-linked agammaglobulinemia. These results have sparked concern regarding whether this drug holds promise in treating norovirus infection, indicating the need for further assessment of the efficacy and working mechanism of nitazoxanide. [...]

Nucleoside analogue 2’-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin

Hepatitis E virus (HEV) infection has emerged as a global health issue, but no approved medication is available. The nucleoside analogue 2’-C-methylcytidine (2CMC), a viral polymerase inhibitor, has been shown to inhibit infection with a variety of viruses, including hepatitis C virus (HCV). Here, we report that 2CMC significantly inhibits the replication of HEV in a subgenomic replication model and in a system using a full-length infectious virus. Importantly, long-term treatment with 2CMC did not result in a loss of antiviral potency, indicating a high barrier to drug resistance development. However, the combination of 2CMC with ribavirin, an off-label treatment for HEV, exerts antagonistic effects. Our results indicate that 2CMC serves as a potential antiviral drug against HEV infection.