Background and Aims: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. Approach and Results: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration–approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. Conclusions: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.

Background and Aims: Chronic HEV infections remain a serious problem in immunocompromised patients, as specifically approved antiviral drugs are unavailable. In 2020, a 24-week multicenter phase II pilot trial was carried out, evaluating the nucleotide analog sofosbuvir by treating nine chronically HEV-infected patients with sofosbuvir (Trial Number NCT03282474). During the study, antiviral therapy reduced virus RNA levels initially but did not lead to a sustained virologic response. Here, we characterize the changes in HEV intrahost populations during sofosbuvir treatment to identify the emergence of treatment-associated variants. Approach and Results: We performed high-throughput sequencing on RNA-dependent RNA polymerase sequences to characterize viral population dynamics in study participants. Subsequently, we used an HEV-based reporter replicon system to investigate sofosbuvir sensitivity in high-frequency variants. Most patients had heterogenous HEV populations, suggesting high adaptability to treatment-related selection pressures. We identified numerous amino acid alterations emerging during treatment and found that the EC₅₀ of patient-derived replicon constructs was up to ~12-fold higher than the wild-type control, suggesting that variants associated with lower drug sensitivity were selected during sofosbuvir treatment. In particular, a single amino acid substitution (A1343V) in the finger domain of ORF1 could reduce susceptibility to sofosbuvir significantly in 8 of 9 patients. Conclusions: In conclusion, viral population dynamics played a critical role during antiviral treatment. High population diversity during sofosbuvir treatment led to the selection of variants (especially A1343V) with lower sensitivity to the drug, uncovering a novel mechanism of resistance-associated variants during sofosbuvir treatment.

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 EC₅₀ 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.

Even 30 years after the discovery of the hepatitis C virus (HCV) in humans there is still no vaccine available. Reasons for this include the high mutation rate of HCV, which allows the virus to escape immune recognition and the absence of an immunocompetent animal model for vaccine development. Phylogenetically distinct hepaciviruses (genus Hepacivirus, family Flaviviridae) have been isolated from diverse species, each with a narrow host range: the equine hepacivirus (EqHV) is the closest known relative of HCV. In this study, we used amplicon-based deep-sequencing to investigate the viral intra-host population composition of the genomic regions encoding the surface glycoproteins E1 and E2. Patterns of E1E2 substitutional evolution were compared in longitudinally sampled EqHV-positive sera of naturally and experimentally infected horses and HCV-positive patients. Intra-host virus diversity was higher in chronically than in acutely infected horses, a pattern which was similar in the HCV-infected patients. However, overall glycoprotein variability was higher in HCV compared to EqHV. Additionally, selection pressure in HCV populations was higher, especially within the N-terminal region of E2, corresponding to the hypervariable region 1 (HVR1) in HCV. An alignment of glycoprotein sequences from diverse hepaciviruses identified the HVR1 as a unique characteristic of HCV: hepaciviruses from non-human species lack this region. Together, these data indicate that EqHV infection of horses could represent a powerful surrogate animal model to gain insights into hepaciviral evolution and HCVs HVR1-mediated immune evasion strategy.

Lung immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for the ability to deal with infection. Using light sheet fluorescence microscopy of hamster lung slices in combination with virological, immunohistochemical, and RNA sequencing analyses, we show a wave of monocyte-derived macrophage (MDM) infiltration and the antiviral response which follows the spread of SARS-CoV-2 through the lungs and lead to virus elimination. These innate immune processes are related to the onset of necrotizing inflammatory and remodeling responses, which manifest as extensive cell death, vascular damage, and cell proliferation. We show that MDM appear in parallel with virus clearance and endothelial injury. Prothrombotic factor upregulation, tissue repair, and alveolar cell proliferation result in tissue remodeling, which is followed by fibrosis despite a decrease in inflammatory and antiviral activities. Although the lung tissue integrity is repaired, longer-term alterations of the lung arise as an outcome of concurrent tissue damage and regeneration processes. IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.