An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes.

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

Targeting cellular cathepsins inhibits hepatitis E virus entry.

BACKGROUND AND AIMS: HEV is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potential of cellular protease during HEV infection. APPROACH AND RESULTS: Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC 50 of ~0.02 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. CONCLUSIONS: In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.

Efficient formation and maintenance of humoral and CD4 T-cell immunity targeting the viral capsid in acute-resolving hepatitis E infection.

BACKGROUND & AIMS: CD4 T cells shape the neutralizing antibody (nAb) response and facilitate viral clearance in various infections. Knowledge of their phenotype, specificity and dynamics in hepatitis E virus (HEV) infection is limited. HEV is enterically transmitted as a naked virus (nHEV) but acquires a host-derived quasi-envelope (eHEV) when budding from cells. While nHEV is composed of the open reading frame (ORF)-2-derived capsid, eHEV particles also contain ORF3-derived proteins. We aimed to longitudinally characterize the HEV-specific CD4 T cells targeting ORF1, 2 and 3 and antibodies against nHEV or eHEV in immunocompetent individuals with acute and resolved HEV infection. METHODS: HEV-specific CD4 T cells were analyzed by intracellular cytokine staining after stimulation with in silico-predicted ORF1- and ORF2-derived epitopes and overlapping peptides spanning the ORF3 region. Ex vivo multiparametric characterization of capsid-specific CD4 T cells was performed using customized MHC class II tetramers. Total and neutralizing antibodies targeting nHEV or eHEV particles were determined. RESULTS: HEV-specific CD4 T-cell frequencies and antibody titers are highest in individuals with acute infection and decline in a time-dependent process with an antigen hierarchy. HEV-specific CD4 T cells strongly target the ORF2-derived capsid and ORF3-specific CD4 T cells are hardly detectable. NAbs targeting nHEV are found in high titers while eHEV particles are less efficiently neutralized. Capsid-specific CD4 T cells undergo memory formation and stepwise contraction, accompanied by dynamic phenotypical and transcriptional changes over time. CONCLUSION: The viral capsid is the main target of HEV-specific CD4 T cells and antibodies in acute-resolving infection, correlating with efficient neutralization of nHEV. Capsid-specific immunity rapidly emerges followed by a stepwise contraction several years after infection. IMPACT AND IMPLICATIONS: The interplay of CD4 T cells and neutralizing antibody responses is critical in the host defense against viral infections, yet little is known about their characteristics in hepatitis E virus (HEV) infection. We conducted a longitudinal study of immunocompetent individuals with acute and resolved HEV infection to understand the characteristics of HEV-specific CD4 T cells and neutralizing antibodies targeting different viral proteins and particles. We found that HEV-specific CD4 T cells mainly target capsid-derived epitopes. This correlates with efficient neutralization of naked virions while quasi-enveloped particles are less susceptible to neutralization. As individuals with pre-existing liver disease and immunocompromised individuals are at risk for fulminant or chronic courses of HEV infection, these individuals might benefit from the development of vaccination strategies which require a detailed knowledge of the composition and longevity of HEV-specific CD4 T-cell and antibody immunity.

Tumour cells can escape antiproliferative pressure by interferon-ß through immunoediting of interferon receptor expression.

Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response, e.g. through a direct impact on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones resistant to the antiproliferative effects of IFNs may contribute to immunoediting of tumours, leading to more aggressive disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that six weeks exposure of cells to IFN-ß in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-ß in a panel of ten different liver cancer cell lines, most prominently in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours. In both, long-term IFN selection and in dedifferentiated tumour cell lines, we found IFNAR2 expression to be substantially reduced, suggesting the receptor complex to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies.

Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic preparedness.

Throughout the SARS-CoV-2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost-effective platform that can be employed in a high-throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS-CoV-2 diagnostics in an academic environment. The strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable with RT-qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.

Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector.

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.

An RNA Interference/Adeno-Associated Virus Vector-Based Combinatorial Gene Therapy Approach Against Hepatitis E Virus.

Hepatitis E virus (HEV) is a major public health problem with limited therapeutic options. Here, we engineered adeno-associated viral vectors of serotype 6 (AAV6) to express short hairpin RNAs (shRNAs) against HEV transcripts with the prospect of down-regulating HEV replication in vivo. We designed 20 different shRNAs, targeting the genome of the HEV genotype 3 (GT3) Kernow-C1 p6 strain, for delivery upon AAV6 transduction. Using an original selectable HEV GT3 reporter replicon, we identified three shRNAs that efficiently down-regulated HEV replication. We further confirmed their inhibitory potency with full-length HEV infection. Seventy-two hours following transduction, HEV replication in both systems decreased by up to 95%. The three most potent inhibitory shRNAs identified were directed against the methyltransferase domain, the junction region between the open reading frames (ORFs), and the 3´ end of ORF2. Targeting all three regions by multiplexing the shRNAs further enhanced their inhibitory potency over a prolonged period of up to 21 days following transduction. Conclusion: Combining RNA interference and AAV vector-based gene therapy has great potential for suppressing HEV replication. Our strategy to target the viral RNA with multiplexed shRNAs should help to counteract viral escape through mutations. Considering the widely documented safety of AAV vector-based gene therapies, our approach is, in principle, amenable to clinical translation.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.

Stem cell-derived polarized hepatocytes.

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Cell Culture Models for Hepatitis E Virus.

Despite a growing awareness, hepatitis E virus (HEV) remains understudied and investigations have been historically hampered by the absence of efficient cell culture systems. As a result, the pathogenesis of HEV infection and basic steps of the HEV life cycle are poorly understood. Major efforts have recently been made through the development of HEV infectious clones and cellular systems that significantly advanced HEV research. Here, we summarize these systems, discussing their advantages and disadvantages for HEV studies. We further capitalize on the need for HEV-permissive polarized cell models to better recapitulate the entire HEV life cycle and transmission.

Stem Cell-Derived Hepatocyte-Like Cells as Model for Viral Hepatitis Research.

Viral hepatitis, the leading cause of liver diseases worldwide, is induced upon infection with hepatotropic viruses, including hepatitis A, B, C, D, and E virus. Due to their obligate intracellular lifestyles, culture systems for efficient viral replication are vital. Although basic and translational research on viral hepatitis has been performed for many years, conventional hepatocellular culture systems are not optimal. These studies have greatly benefited from recent efforts on improving cell culture models for virus replication and infection studies. Here we summarize the use of human stem cell-derived hepatocyte-like cells for hepatotropic virus infection studies, including the dissection of virus-host interactions and virus-induced pathogenesis as well as the identification and validation of novel antiviral agents.

Stem Cell-Derived Culture Models of Hepatitis E Virus Infection.

Similar to other hepatotropic viruses, hepatitis E virus (HEV) has been notoriously difficult to propagate in cell culture, limiting studies to unravel its biology. Recently, major advances have been made by passaging primary HEV isolates and selecting variants that replicate efficiently in carcinoma cells. These adaptations, however, can alter HEV biology. We have explored human embryonic or induced pluripotent stem cell (hESC/iPSC)-derived hepatocyte-like cells (HLCs) as an alternative to conventional hepatoma and hepatocyte cell culture systems for HEV studies. HLCs are permissive for nonadapted HEV isolate genotypes (gt)1-4 replication and can be readily genetically manipulated. HLCs, therefore, enable studies of pan-genotype HEV biology and will serve as a platform for testing anti-HEV treatments. Finally, we discuss how hepatocyte polarity is likely an important factor in the maturation and spread of infectious HEV particles.

Embryonic or Induced Pluripotent Stem Cell-Derived Hepatocellular Systems for HCV Culture.

Human-induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) constitute a powerful tool for modeling hepatotropic pathogen infections in cell culture. Meanwhile, CRISPR-Cas9 technology enables precise editing of stem cell genomes to generate patient-specific disease models and thus development of personalized experimental systems. Here we present a detailed stepwise protocol for the differentiation of stem cells to hepatocyte-like cells for HCV studies in cell culture. We also outline the use of an inducible iCRISPR platform for the rapid and efficient modification of host factors of interest to better understand their function during HCV infection.

Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion.

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.

The natural compound silvestrol inhibits hepatitis E virus (HEV) replication in vitro and in vivo.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients and type I interferon (IFN) has been evaluated in a few infected transplantation patients in vivo. However, no effective and specific treatments against HEV infections are currently available. In this study, we evaluated the natural compound silvestrol, isolated from the plant Aglaia foveolata, and known for its specific inhibition of the DEAD-box RNA helicase eIF4A in state-of-the-art HEV experimental model systems. Silvestrol blocked HEV replication of different subgenomic replicons in a dose-dependent manner at low nanomolar concentrations and acted additive to ribavirin (RBV). In addition, HEV p6-based full length replication and production of infectious particles was reduced in the presence of silvestrol. A pangenotypic effect of the compound was further demonstrated with primary isolates from four different human genotypes in HEV infection experiments of hepatocyte-like cells derived from human embryonic and induced pluripotent stem cells. In vivo, HEV RNA levels rapidly declined in the feces of treated mice while no effect was observed in the vehicle treated control animals. In conclusion, silvestrol could be identified as pangenotypic HEV replication inhibitor in vitro with additive effect to RBV and further demonstrated high potency in vivo. The compound therefore may be considered in future treatment strategies of chronic hepatitis E in immunocompromised patients.

Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown.

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such as double-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.

Hepatitis E virus replication and interferon responses in human placental cells.

Hepatitis E virus (HEV) is a member of the genus Orthohepevirus in the family Hepeviridae and the causative agent of hepatitis E in humans. HEV is a major health problem in developing countries, causing mortality rates up to 25% in pregnant women. However, these cases are mainly reported for HEV genotype (gt)1, while gt3 infections are usually associated with subclinical courses of disease. The pathogenic mechanisms of adverse maternal and fetal outcome during pregnancy in HEV-infected pregnant women remain elusive. In this study, we observed that HEV is capable of completing the full viral life cycle in placental-derived cells (JEG-3). Following transfection of JEG-3 cells, HEV replication of both HEV gts could be observed. Furthermore, determination of extracellular and intracellular viral capsid levels, infectivity, and biophysical properties revealed production of HEV infectious particles with similar characteristics as in liver-derived cells. Viral entry was analyzed by infection of target cells and detection of either viral RNA or staining for viral capsid protein by immunofluorescence. HEV gt1 and gt3 were efficiently inhibited by ribavirin in placental as well as in human hepatoma cells. In contrast, interferon-α sensitivity was lower in the placental cells compared to liver cells for gt1 but not gt3 HEV. Simultaneous determination of interferon-stimulated gene expression levels demonstrated an efficient HEV-dependent restriction in JEG-3. Conclusion: We showed differential tissue-specific host responses to HEV genotypes, adding to our understanding of the mechanisms contributing to fatal outcomes of HEV infections during pregnancy. Using this cell-culture system, new therapeutic options for HEV during pregnancy can be identified and evaluated. (Hepatology Communications 2018;2:173-187).

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

BACKGROUND & AIMS: The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. METHODS: Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. RESULTS: HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. CONCLUSIONS: Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.

Intrinsic Immunity Shapes Viral Resistance of Stem Cells.

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.