The hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. RNA viruses like HEV can establish viral populations with high intra-host variability, enabling them to rapidly adapt to changing immune responses, as observed in chronic infections. This study aimed to investigate how intra-host evolution shapes HEV populations during acute asymptomatic infection. Using a highly sensitive HEV amplicon sequencing approach, we characterized intra-host viral diversity and mutational signatures present in serum samples from a cohort of over 80 asymptomatic blood donors infected with acute HEV-3. The overall diversity within the host was constrained; however, several recurrent substitutions were identified, and four mutations in the polymerase region were found to be enriched among the donors. Despite being markedly replication-deficient in vitro, their replication defects could be rescued in an RNA-dependent RNA polymerase trans-complementation assay. Longitudinal sampling in a subset of donors revealed temporal shifts in variant frequencies, indicating ongoing early selection dynamics. In contrast to chronic HEV infection in immunosuppressed patients, acute-phase populations exhibited a significantly lower number of single-nucleotide variants (SNVs) and an absence of high-frequency variants. The presence of premature stop codons and other defective genomes was primarily detected during the acute phase of infection. This finding further supports the hypothesis that the early evolutionary landscape is highly dynamic, but constrained. Although HEV diversity is markedly restricted during acute infection, early evolutionary changes indicate that selective forces act even within the short window of acute, self-limiting, and asymptomatic disease. These findings offer mechanistic insights into early intra-host evolution, highlight conditions under which deleterious variants can transiently persist, and lay the groundwork for linking genotypic features to clinical outcomes and treatment responsiveness.IMPORTANCEThe hepatitis E virus is the leading cause of acute viral hepatitis globally. While studies of chronic infections have shown that HEV can evolve and adapt in ways that influence antiviral treatment responses, little is known about how the virus changes during the early, acute phase of infection. By analyzing viral populations in asymptomatic blood donors, this study demonstrates that HEV undergoes dynamic evolutionary changes even during short, self-limiting infections. Although overall viral diversity remains restricted, selective pressures still drive the emergence of new variants, including some with impaired replication capacity. These findings provide important mechanistic insight into early intra-host viral evolution and the conditions that allow defective variants to transiently persist. This work establishes a foundation for future studies linking viral genetic variation to disease progression, clinical outcomes, and treatment responsiveness.

BACKGROUND AND AIMS:Hepatitis E virus (HEV) infection is very frequent in Europe with more than 2 million annual infections. Patients with liver cirrhosis may face an increased risk of suffering from acute-on-chronic liver failure (ACLF) due to HEV infection. We explored the consequences and prevalence of HEV infection in individuals with liver cirrhosis. METHODS:We retrospectively analysed the clinical outcome of all consecutive patients who were hospitalized at our center due to acute HEV infection and analysed their outcome between 2014 and 2024. Next, we tested 249 sera from 184 cirrhotic patients during individual episodes of acute hepatic decompensation for anti-HEV IgM and HEV-RNA to analyse the relevance of acute HEV infection as a triggering event. Finally, we established a single center cohort of patients with advanced liver cirrhosis, and assessed the anti-HEV IgG seroprevalence (n = 332). RESULTS:Over the past decade, 32 patients with liver cirrhosis who were hospitalized due to acute HEV infection were identified. Among these patients, 16 (50%) developed ACLF, resulting in five fatalities (31.3%) and three individuals (18.8%) requiring liver transplantation for survival. Of 249 sera obtained during acute hepatic decompensation, 11 (4.4%) were either HEV-RNA positive (n = 2) and/or anti-HEV IgM positive (n = 10), linking HEV infection to these acute decompensations. Screening of patients with liver cirrhosis for anti-HEV IgG showed that 67.2% of patients (223/332) were anti-HEV negative and thus at potential risk for future HEV infection. CONCLUSIONS:Patients with advanced liver cirrhosis are at risk of acute HEV infection, which is a relevant cause of hepatic decompensation and ACLF with high mortality in these patients. TRIAL REGISTRATION:DRKS00010664; NCT04801290.

Background: Alpha-1 antitrypsin deficiency (AATD) caused by the PI*ZZ mutation (Glu342Lys) results in hepatic accumulation of misfolded AAT-Z protein and reduced circulating AAT levels, leading to progressive liver disease and emphysema. Gene correction therapy represents a potentially curative approach by directly correcting the underlying genetic defect. We report the first case of successful hepatic gene correction with early histological and functional assessment. Methods/Case presentation: We report the case of a 66-year-old male patient with PI*ZZ AATD who underwent gene correction therapy within the YOLT-202 phase I/Ia clinical trial (clinical trial.gov ID NCT07193615). Ten weeks post treatment a liver biopsy was performed to re-evaluate pre-existing F2 liver fibrosis as measured by elastography before entering the study. Serum samples allowed functional assessment of the AAT-mediated elastase inhibition. Results: Liver biopsy did not show signs of hepatic inflammation and demonstrated 54% (Sanger) and 57% (Illumina) gene correction rate of the PI*ZZ variant on the DNA level with no bystander edits or off-target effects. Following a transient elevation of transaminases during the early post-treatment period, liver enzymes normalized. Monthly serum AAT measurements demonstrated biologically active and stable therapeutic levels throughout follow-up. Conclusions: This case demonstrates efficient and precise hepatic gene correction without concerning histological alterations and with substantial improvement of functional parameters, supporting the feasibility and safety of gene editing approaches for AATD.

The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Although most infections are self-limiting, zoonotic genotypes can persist in immunocompromised individuals. Transmitted via the fecal-oral route, HEV has been suggested to directly infect the intestinal epithelium, a tissue with high regenerative capacity. Here, we demonstrate that HEV predominantly infects proliferative transit-amplifying and intestinal stem cells within the crypts of human pluripotent stem cell-derived intestinal organoids (hIOs). Supporting this, we detected HEV RNA in the intestinal crypts of an HEV-infected patient. We further found that HEV infection spreads through cell division and is maintained in hIOs for more than 40 days, contrasting with acute hepatitis A virus, whose infections are rapidly cleared from hIOs. Given the self-renewal ability and metabolic constraints of proliferative intestinal progenitor cells, our findings suggest that intestinal crypts could serve as reservoirs for chronic HEV infection and highlight the intestinal crypt as a primary target for viral infection in the gastrointestinal tract.

INTRODUCTION:The hepatitis E virus (HEV; species Paslahepevirus balayani) is a common human pathogenic and zoonotic virus that can cause both acute fulminant and chronic hepatitis. Despite its reputation as a hepatotropic virus, HEV infection is also associated with a number of extrahepatic diseases, including kidney disorders. However, the extent to which HEV replicates in kidney cells remains unclear. The present study aims to investigate the capacity of HEV to propagate in kidney cells in vitro and to assess whether HEV displays mutational signatures that correlate with compartmentalisation in vivo. METHODS:We use HEV cell culture models to study the replication cycle and the effect of antivirals in human kidney cell lines and primary cells. In addition, we identified patients with chronic HEV infection (n = 9) from which we then sequenced the viral RNA of urine, stools and plasma to analyse the viral sequence composition, to assess intra-host diversity and compartmentalisation (n = 2). RESULTS:A wide range of human kidney cell lines as well as primary cells supports viral entry, replication and propagation of HEV in vitro. Interestingly, the broad-spectrum antiviral ribavirin was less effective in inhibiting HEV replication in some kidney cells. Sequencing of HEV RNA-directed RNA polymerase coding region from plasma, stool and urine and subsequent phylogenetic analysis revealed diversification of HEV into tissue-specific viral subpopulations. In particular, the viruses derived from urine were found to be distinct from those derived from plasma and stool. CONCLUSIONS:In conclusion, kidney cells support the propagation of HEV in vitro and exhibit reduced sensitivity to antiviral treatment. Furthermore, HEV patient-derived sequences demonstrated compartmentalisation into distinct clusters that correlated with sample source. Collectively, these data indicate the potential for extrahepatic replication of HEV, which may result in clinically significant disease or serve as a reservoir for patient relapse. TRIAL REGISTRATION:HepNet-SofE study (NCT03282474).

Hepatitis E virus (HEV) poses a significant risk to human health. In Europe, the majority of HEV infection are caused by the zoonotic genotype 3 (HEV-3), which can cause chronic hepatitis E in immunocompromised patients and those with pre-existing liver disease, and may eventually develop into fatal liver cirrhosis. In this study, we examined the effectiveness of a monoclonal antibody (MAb) treatment strategy using a well established HEV-3 pig model with intravenous infection. For this purpose, nine MAbs raised against the viral capsid protein were generated and the neutralizing activities were compared using in vitro assays. The antibody with the highest neutralizing activity, MAb 5F6A1, was selected for an in vivo study in pigs infected with HEV-3. Following the initial infection of pigs with HEV-3, MAb 5F6A1 was administered intravenously one and seven days post-infection. The results suggest MAb 5F6A1 significantly reduced viremia and virus shedding in pigs infected with HEV-3. This study provides significant insight into the dynamics of HEV infection in pigs and highlights the efficacy of MAb based therapy as an option for treating HEV in porcine hosts and, potentially, humans.

BACKGROUND AND AIMS:Hepatitis E virus (HEV) poses a significant global health concern, with millions of annual infections and a notable impact on public health. Although HEV is the leading cause of acute viral hepatitis worldwide, there is a substantial lack of approved and licensed vaccines. In this study, we evaluated the efficacy of several protein- and DNA-based vaccine candidates against HEV using a combined in vitro/in vivo workflow. METHODS:Corresponding vaccine candidates were produced, biochemically analysed and characterised. The general immunogenicity of suitable vaccine candidates was initially evaluated in a rabbit model. Resulting antibodies were assessed for their reactivity and neutralising efficiency. Finally, the most effective candidates were tested in a pig infection model using a prime-boost vaccination regimen. RESULTS:Using this approach, we analysed a total of seven vaccine candidates and demonstrated that the two most promising candidates significantly reduced virus shedding in swine faecal samples after infection. However, no sterile immunity was achieved. CONCLUSIONS:This study conducted a comprehensive analysis to establish a rational approach for post-vaccination immune responses in pigs. The insights gained from this research are expected to significantly contribute to the development and evaluation of future vaccine candidates for pig herds, ultimately reducing viral dissemination among pigs and preventing HEV transmission from pigs to humans. These findings hold important translational value, offering a foundation for both improving animal health and safeguarding public health.

Background and Aims: Hepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems. Methods: We exploited transposon-mediated random insertion and selection of viable clones to identify sites in the HEV open reading frame 2 (ORF2) protein, corresponding to the viral capsid, allowing for the insertion of reporter sequences in a functional context. Results: Short sequence insertions (5 amino acids) were tolerated at four distinct sites in the C-terminal region of the ORF2 protein, without significantly affecting viral capsid expression and subcellular localization as well as virus production. Full-length HEV genomes harboring larger sequence insertions such as an HA epitope tag, a highly sensitive miniaturized luciferase reporter (HiBiT) or a split GFP at these sites conserved their ability to produce infectious virus, with about a 1-log decrease in viral titers. Findings were confirmed in two different HEV genotype 3 clones. In addition, we demonstrate that HiBiT-tagged HEV, offering rapid and several-log amplitude detection, can be used for the evaluation of antiviral drugs and neutralizing antibodies. Conclusions: We describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture. Impact and implications:: Hepatitis E virus infection is one of the most frequent causes of acute hepatitis and jaundice worldwide. As treatment options are limited and a vaccine is not universally available, the development of molecular tools to facilitate the identification of new therapeutic strategies is crucial. Based on a screening approach to identify viable insertion sites in the viral genome, we describe a versatile system for preparing recombinant viruses harboring split-reporter tags, i.e. luciferase and GFP. Proof-of-concept experiments revealed that convenient and quantitative monitoring of viral infection and replication is possible with this system, allowing for the evaluation of antiviral drugs and neutralizing antibodies.