Targeting cellular cathepsins inhibits hepatitis E virus entry.

BACKGROUND AND AIMS: The hepatitis E virus (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 potentials of cellular proteases 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 ~ 0.01 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.

Dynamic evolution of the sofosbuvir-associated variant A1343V in HEV-infected patients under concomitant sofosbuvir-ribavirin treatment.

Background & Aims: In the absence of a hepatitis E virus (HEV)-specific antiviral treatment, sofosbuvir has recently been shown to have antiviral activity against HEV in vivo. However, a variant, A1343V, that is strongly associated with viral relapse impedes treatment success. In this study, we investigated the occurrence of variants during sofosbuvir and ribavirin treatment in vivo and assessed the sensitivity of resistance-associated variants to concurrent treatment in cell culture. Methods: Two patients with chronic HEV infection that did not clear infection under ribavirin treatment were subsequently treated with a combination of sofosbuvir and ribavirin. We determined response to treatment by measuring liver enzymes and viral load in blood and stool. Moreover, we analyzed viral evolution using polymerase-targeted high-throughput sequencing and assessed replication fitness of resistance-associated variants using a HEV replicon system. Results: Combination treatment was successful in decreasing viral load towards the limit of quantification. However, during treatment sustained virological response was not achieved. Variants associated with sofosbuvir or ribavirin treatment emerged during treatment, including A1343V and G1634R. Moreover, A1343V, as a single or double mutation with G1634R, was associated with sofosbuvir resistance during concomitant treatment in vitro. Conclusions: These results highlight the importance of variant profiling during antiviral treatment of patients with chronic infection. Understanding how intra-host viral evolution impedes treatment success will help guide the design of next-generation antivirals. Impact and implications: The lack of hepatitis E virus (HEV)-specific antivirals to treat chronic infection remains a serious health burden. Although ribavirin, interferon and sofosbuvir have been reported as anti-HEV drugs, not all patients are eligible for treatment or clear infection, since resistant-associated variants can rapidly emerge. In this study, we analyzed the efficacy of sofosbuvir and ribavirin combination treatment in terms of HEV suppression, the emergence of resistance-associated variants and their ability to escape treatment inhibition in vitro. Our results provide novel insights into evolutionary dynamics of HEV during treatment and thus will help guide the design of next-generation antivirals.

Host determinants and responses underlying SARS-CoV-2 liver tropism.

Hepatic sequelae are frequently reported in coronavirus disease 2019 cases and are correlated with increased disease severity. Therefore, a detailed exploration of host factors contributing to hepatic impairment and ultimately infection outcomes in patients is essential for improved clinical management. The causes of hepatic injury are not limited to drug-mediated toxicity or aberrant host inflammatory responses. Indeed, multiple studies report the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in liver autopsies and the susceptibility of explanted human hepatocytes to infection. In this review, we confirm that hepatic cells express an extensive range of factors implicated in SARS-CoV-2 entry. We also provide an overview of studies reporting evidence for direct infection of liver cell types and the infection-induced cell-intrinsic processes that likely contribute to hepatic impairment.

Halogenated Rocaglate Derivatives: Pan-antiviral Agents against Hepatitis E Virus and Emerging Viruses.

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4', 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Janus kinase-inhibition modulates hepatitis E virus infection.

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.

Emergence of resistance-associated variants during sofosbuvir treatment in chronically infected hepatitis E patients.

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 EC 50 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.

Hepatitis E virus species barriers: seeking viral and host determinants.

The intimate relationship between virus and host cell can result in highly adapted viruses that are restricted to a single host. However, some viruses have the ability to infect multiple host species. Remarkably, hepatitis E viruses (HEV) comprise genotypes that are either 'single-host' or 'multi-host' genotypes, a trait that raises fundamental questions: Why do different genotypes differ in their host range, despite a high degree of genomic similarity? What are the underlying molecular determinants that shape species barriers? Here, we review the current knowledge of viral and host determinants that may affect the evolutionary trajectories of HEV. We also provide a perspective on techniques and methods that address open questions of HEV host range and adaptation.

Identification of structurally re-engineered rocaglates as inhibitors against hepatitis E virus replication.

Hepatitis E virus (HEV) infections are a leading cause of acute viral hepatitis in humans and pose a considerable threat to public health. Current standard of care treatment is limited to the off-label use of nucleoside-analog ribavirin (RBV) and PEGylated interferon-α, both of which are associated with significant side effects and provide limited efficacy. In the past few years, a promising natural product compound class of eukaryotic initiation factor 4A (eIF4A) inhibitors (translation initiation inhibitors), called rocaglates, were identified as antiviral agents against RNA virus infections. In the present study, we evaluated a total of 205 synthetic rocaglate derivatives from the BU-CMD compound library for their antiviral properties against HEV. At least eleven compounds showed inhibitory activities against the HEV genotype 3 (HEV-3) subgenomic replicon below 30 nM (EC50 value) as determined by Gaussia luciferase assay. Three amidino-rocaglates (ADRs) (CMLD012073, CMLD012118, and CMLD012612) possessed antiviral activity against HEV with EC50 values between 1 and 9 nM. In addition, these three selected compounds inhibited subgenomic replicons of different genotypes (HEV-1 [Sar55], wild boar HEV-3 [83-2] and human HEV-3 [p6]) in a dose-dependent manner and at low nanomolar concentrations. Furthermore, tested ADRs tend to be better tolerated in primary hepatocytes than hepatoma cancer cell lines and combination treatment of CMLD012118 with RBV and interferon-α (IFN-α) showed that CMLD012118 acts additive to RBV and IFN-α treatment. In conclusion, our results indicate that ADRs, especially CMLD012073, CMLD012118, and CMLD012612 may prove to be potential therapeutic candidates for the treatment of HEV infections and may contribute to the discovery of pan-genotypic inhibitors in the future.

In-depth analysis of T cell immunity and antibody responses in heterologous prime-boost-boost vaccine regimens against SARS-CoV-2 and Omicron variant.

With the emergence of novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs), vaccination studies that elucidate the efficiency and effectiveness of a vaccination campaign are critical to assess the durability and the protective immunity provided by vaccines. SARS-CoV-2 vaccines have been found to induce robust humoral and cell-mediated immunity in individuals vaccinated with homologous vaccination regimens. Recent studies also suggest improved immune response against SARS-CoV-2 when heterologous vaccination strategies are employed. Yet, few data exist on the extent to which heterologous prime-boost-boost vaccinations with two different vaccine platforms have an impact on the T cell-mediated immune responses with a special emphasis on the currently dominantly circulating Omicron strain. In this study, we collected serum and peripheral blood mononuclear cells (PBMCs) from 57 study participants of median 35-year old's working in the health care field, who have received different vaccination regimens. Neutralization assays revealed robust but decreased neutralization of Omicron VOC, including BA.1 and BA.4/5, compared to WT SARS-CoV-2 in all vaccine groups and increased WT SARS-CoV-2 binding and neutralizing antibodies titers in homologous mRNA prime-boost-boost study participants. By investigating cytokine production, we found that homologous and heterologous prime-boost-boost-vaccination induces a robust cytokine response of CD4+ and CD8+ T cells. Collectively, our results indicate robust humoral and T cell mediated immunity against Omicron in homologous and heterologous prime-boost-boost vaccinated study participants, which might serve as a guide for policy decisions.

Beyond the Usual Suspects: Hepatitis E Virus and Its Implications in Hepatocellular Carcinoma.

Hepatitis E virus infections are the leading cause of viral hepatitis in humans, contributing to an estimated 3.3 million symptomatic cases and almost 44,000 deaths annually. Recently, HEV infections have been found to result in chronic liver infection and cirrhosis in severely immunocompromised patients, suggesting the possibility of HEV-induced hepatocarcinogenesis. While HEV-associated formation of HCC has rarely been reported, the expansion of HEV's clinical spectrum and the increasing evidence of chronic HEV infections raise questions about the connection between HEV and HCC. The present review summarizes current clinical evidence of the relationship between HEV and HCC and discusses mechanisms of virus-induced HCC development with regard to HEV pathogenesis. We further elucidate why the development of HEV-induced hepatocellular carcinoma has so rarely been observed and provide an outlook on possible experimental set-ups to study the relationship between HEV and HCC formation.

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

Antiviral Effect of Budesonide against SARS-CoV-2.

Treatment options for COVID-19, a disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, are currently severely limited. Therefore, antiviral drugs that efficiently reduce SARS-CoV-2 replication or alleviate COVID-19 symptoms are urgently needed. Inhaled glucocorticoids are currently being discussed in the context of treatment for COVID-19, partly based on a previous study that reported reduced recovery times in cases of mild COVID-19 after inhalative administration of the glucocorticoid budesonide. Given various reports that describe the potential antiviral activity of glucocorticoids against respiratory viruses, we aimed to analyze a potential antiviral activity of budesonide against SARS-CoV-2 and circulating variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta). We demonstrate a dose-dependent inhibition of SARS-CoV-2 that was comparable between all viral variants tested while cell viability remains unaffected. Our results are encouraging as they could indicate a multimodal mode of action of budesonide against SARS-CoV-2 and COVID-19, which could contribute to an improved clinical performance.

Clinical Course of Infection and Cross-Species Detection of Equine Parvovirus-Hepatitis.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler's disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler's disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013-2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013-2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.

In Vitro Lung Models and Their Application to Study SARS-CoV-2 Pathogenesis and Disease.

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic.

Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p.

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks.

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.