Update on Hepatitis E Virus Infection 2025: Insights From an International Symposium

2.1. Potential Reservoir of HEV Infection in the Intestinal Stem Cell Niche (By Dr. Viet Loan Dao Thi)

As HEV is transmitted enterically and is often ingested via contaminated food or water, it has to overcome the intestinal barrier in order to reach the liver, which is the main replication site of the virus [1, 8]. Up to now, it is not clear if HEV can infect the intestinal barrier or if it simply passes it via transcytosis or other modes of translocation. Two studies already suggested that HEV can replicate in intestinal cells [10, 11], which motivated the group of Dr. Dao Thi to investigate this potential replication site further. To do so, the group established intestinal organoids (hIOs), derived from induced pluripotent stem cells (Figure 1). These organoids represent the cell heterogenicity of the human intestine and have a stem cell crypt containing proliferating cells. The group was able to infect hIOs with cell culture‐ and patient‐derived HEV and showed that the infection is primarily maintained in the crypts of organoids (Figure 1). This viral replication in the intestinal crypts was also found in intestinal biopsies from HEV patients. Compared to HAV, which was cleared from the hIOs within 4 days, HEV was able to persist in hIOs for up to 40 days, likely through cell division‐mediated spread. Dr. Dao Thi concluded that the intestine should be regarded as a potential reservoir for HEV infection and this should be considered in the development of future therapeutics.

2.2. Zoonotic Potential of Rat Hepatitis E Virus in Germany (By Prof. Dr. Reimar Johne)

While HEV1 and HEV2 only infect humans and are transmitted via the faecal‐oral route, for example due to the consumption of contaminated water, other HEV strains can infect humans via zoonotic infections. It is broadly known that HEV3 and HEV4 infect domestic pigs and wild boars and can be transmitted to humans via the consumption of contaminated meat products [12]. In the early 2000s it has been suspected that rats can also be infected with HEV. In 2010, Reimar Johne and colleagues could show that distinct HEV strains are circulating in rats in Hamburg, which are only distantly related to the HEV‐1–4 genotypes and rather cluster in a separate group [13, 14]. During the following years, this virus was found in rats all over the world, and the phylogenetic analysis led to the classification of rat HEV as a separate species Rocahepevirus ratti (HEV‐C) [15, 16, 17]. Since the identification of the virus, it has been identified globally in rats, with most reports from Asian and European countries. Two distinct genotypes of the virus have been described, termed HEV‐C1 and ‐C2, which occur in different host species [15, 18]. Since 2018, several human rat HEV infections, most of which occurred in Hongkong and Spain, and often connected with symptomatic hepatitis, have been described [19, 20, 21]. This motivated Prof. Johne to continue his research on rat HEV in Germany. The human cell line Huh7‐Lunet‐BLR is susceptible to replication of rat HEV with no obvious differences in the replication of strains isolated from rat and human of Germany and Hongkong, indicating a general zoonotic potential of rat HEV strains [22]. After sampling wild rats in Berlin in 2023, rat HEV could be identified in 8.4% of the specimens. This number was similar to the 11.5% of rats in Berlin, which were tested positive for rat HEV in 2009/2010, indicating continued circulation of the virus in this wild rat population. In an analysis of 1000 human samples from the Charité—Universitätsmedizin Berlin, one of the analysed samples was positive for rat HEV RNA, which proved that human infections of rat HEV also occur in Germany although with a low detection rate (0.1%) [23]. Taken together, rat HEV continuously circulates in wild rats, which represent a permanent source of infection for humans, even though infections obviously occur at a low incidence in Germany.

2.3. RNA Interference Strategies Targeting HEV—Combining RNA Interference and RIG‐I Activation to Inhibit Hepatitis E Virus Replication (By Mathias Ziersch)

RNA‐Interference (RNAi) is the process in which short, double‐stranded RNA segments (siRNA) interact with the Ago2 protein to form the RISC complex, which targets RNA complementary to the siRNA and cleaves it [24]. There are several reasons why the utilisation of RNAi can be considered as a treatment option for HEV. Firstly, siRNA can be modified with N‐Acetylgalactosamin, which binds to the asiaglycoprotein receptor. This receptor mediates endocytosis in liver cells, especially under cirrhotic conditions. Therefore, the siRNA is delivered to the liver efficiently [25]. Additionally, phase II studies have already shown successes in treating a Hepatitis B virus infection with siRNA [26, 27]. Mathias Ziersch decided on different siRNAs, targeting either ORF3 of the HEV RNA, as this is a very conserved region, or the viral helicase, as a control, to test for their anti‐HEV capacity. Two of the three siRNAs targeting ORF3 efficiently reduced HEV RNA copy numbers in persistently and acutely infected cell lines. The optimal effect could be observed 96 h after a dual siRNA transfection within 24 h. The reduction of the HEV RNA was dose‐dependent and already occurred at very low doses in the sub‐nanomolar range (Figure 2) [28]. To improve the anti‐viral effect, a 5′‐triphosphate modification was covalently coupled to the siRNA to activate RIG‐I, a cytoplasmic pattern recognition receptor that recognises viral RNA. It was demonstrated that induction of RIG‐I effectively suppresses HEV replication [29]. Although the dual‐action strategy, combining activation of the adaptive immune response with stimulation of the intrinsic RNAi pathway, did not yield additive effects, this approach may help prevent the emergence of escape mutants resistant to siRNA‐mediated silencing (Figure 2). In the future, the group wants to test 3D organ models to evaluate the performance of the siRNA against HEV [30] and they want to combine their siRNA treatment with the administration of ribavirin derivates to test the antiviral potential of combination therapy.

2.4. Dynamics of Hepatitis E Virus Variant Evolution During Ribavirin Treatment of Chronic Infection: An In‐Depth In Vitro Versus In Vivo Comparison (By Dr. Dominik Harms)

Chronic HEV infection may manifest in immunocompromised patients, indicated by the persistence of HEV RNA in blood or stool for more than 3 months. As there is currently no specific therapy for HEV, treatment options are limited to off‐label administration of ribavirin [7, 31]. However, past studies have described selection of viral variants under ribavirin administration that are associated with treatment failure [32]. Dr. Harms presented the case of a kidney transplant patient, suffering from chronic HEV infection since 2009. The patient was repeatetly treated with ribavirin, achieving no sustained virological response. During treatment, Sanger sequencing of the HEV polymerase region was performed multiple times, demonstrating the emergence of multiple ribavirin‐associated viral variants. Known and previously unreported variants were identified by deep sequencing of the complete viral genome at time points before and after treatment discontinuation.

To further analyse the evolutionary dynamics of ribavirin‐associated variants, Dr. Harms used a cell line persistently infected with an HEV isolate from the presented patient [33]. After multiple rounds of antiviral treatment, viral response to ribavirin declined and specific viral variants became dominant within the quasispecies. By comparing patient‐ and culture‐specific variants, several common ORF1 and ORF2 mutations were identified. Some of these variants have already been described in the context of ribavirin while others were newly identified in this study.

Dr. Harms concluded that the periodical ribavirin treatment promotes the emergence and selection of treatment failure‐associated viral variants and therefore a high‐dose prolonged treatment might be beneficial. Additionally, one should consider the impact of other viral variants in the context of treatment failure other than those that are already known.

2.5. Round Table Discussion: Research in HEV—Key Challenges and Future Perspectives

Following each session of scientific presentations, a round table discussion provided an opportunity for interactive dialogue, critical feedback, and interdisciplinary exchange.

In the course of the discussion, various challenges regarding HEV were debated, which are presented here in summary.

3.1. The Hepatitis E Virus—More Than Just the 5th Wheel on the Wagon (By Prof. Dr. Jörg Hofmann)

As the number of reported HEV cases is continuously increasing in Germany, there is a great need for reliable diagnostic tools for HEV [49]. Especially, the detection of chronic HEV cases presents clinical challenges as the time of the appearance of first antibodies can vary, liver enzymes are only moderately elevated, and IgM can often be detected for a very long time period [7]. In a comparative analysis, tests for the detection of HEV IgM as well as IgG antibodies performed well in terms of specificity and sensitivity [50]. Nevertheless, factors like the timing of sample collection, the patient's immune status, and the HEV genotype can influence test performance. Therefore, the European Association for the Study of the Liver (EASL) recommends the combination of testing for anti‐HEV antibodies as well as HEV nucleic acid to detect HEV infections [7]. Meanwhile, there is no point‐of‐care test approved by the FDA or prequalified by the WHO, and therefore the establishment of these tests is required by each institution separately [51].

The Labor Berlin analysed samples from more than 15 000 patients in order to evaluate clinical testing of HEV for acutely and chronically infected patients. They found that regarding IgG and HEV RNA levels, but not IgM levels, there were significantly more male individuals that tested positive. Furthermore, they found that the HEV viral load is significantly higher in chronically infected individuals than in acutely infected patients. Based on this data, the Labor Berlin comprised recommendations to identify acute and chronic HEV infections in immunocompetent and immunocompromised patients for their sender clientele (Table 1). Lastly, Prof. Hofmann highlighted that it is not sufficient to analyse transaminase levels for viral monitoring, as these liver values are reduced by a Ribavirin treatment, even if the viral loads are still high.

3.2. A Novel Class of Human Monoclonal Antibodies Neutralising HEV (By Dr. Katja Dinkelborg)

HEV infections are the most common cause of acute viral hepatitis and can lead to severe outcomes in patients with risk factors like pregnancy or an underlying liver disease [2, 6, 52]. Nevertheless, the therapeutic options are limited to the off‐label use of Ribavirin and therefore it is important to find direct antivirals to treat HEV infections. In collaboration with Dr. Krey from the University of Lübeck, Dr. Dinkelborg and the research group of Dr. Patrick Behrendt isolated memory B‐cells binding the HEV capsid from convalescent HEV patients. These human antibodies were tested for their HEV binding and neutralisation. In cell culture experiments, the antibody candidates neutralised non‐enveloped HEV particles with different efficiencies, with some also displaying a neutralising effect against the pseudo‐enveloped virus (Figure 3) [53]. An important consideration when designing neutralising antibodies against HEV is that the ORF2 protein, which forms the capsid of the virus, occurs in a glycosylated form. This glycosylated ORF2 protein is likely shed by infected cells to capture anti‐HEV antibodies and therefore avoid virus neutralisation [54, 55]. Of the different antibody candidates, there were at least two glycan‐sensitive antibodies that only bound the infectious ORF2 protein but not the glycosylated form of the protein. These glycan‐sensitive antibodies were able to neutralise HEV even in the presence of high amounts of the glycosylated ORF2 (Figure 3).

As the tested antibodies bind to a conserved domain of the viral protein, the glycan‐sensitive antibodies also readily neutralise HEV1–4 as well as rat HEV. These antibodies also protect animals in different infection settings from an HEV infection.

In the future, these neutralising antibodies could potentially present a therapeutic option for HEV infections and serve as a useful tool in HEV research [53].

3.3. Novel Antivirals to Combat HEV Infections (By Dr. Mara Klöhn)

Several compounds have been identified as potential inhibitors of HEV infection. These compounds target various stages of the viral life cycle; however, most of them are confined to basic research and have yet to progress to pre‐clinical studies. Consequently, there is demand for new antiviral agents (Figure 4) [56].

Dr. Klöhn and colleagues discovered that the cathepsin inhibitor K11777 blocks the cleavage of the HEV capsid protein by cathepsins during virus infection, thereby demonstrating antiviral activity against HEV. This inhibitor effectively reduced HEV infection in both hepatoma cells and primary human hepatocytes [57].

In cooperation with KU Leuven, Janssen Pharmaceuticals and Ruhr University Bochum, researchers identified an HEV polymerase inhibitor (JNJ‐9117), which was previously recognised for inhibiting Hepatitis C virus replication. JNJ‐9117 inhibited HEV replication in hepatic and extrahepatic cell lines as well as in primary human hepatocytes. Most strikingly, the inhibitor had a pronounced antiviral effect against HEV in rats, both as prophylactic and therapeutic treatment [58].

Furthermore, Dr. Klöhn presented data on antiviral compound screens consisting of libraries of FDA‐approved compounds and nucleoside/nucleotide analogues to find potential antiviral agents against HEV. This screening led to several promising studies. Notably, the inhibition of the proteins METTL3 or FTO, which are involved in the RNA methylation and demethylation pathways, was found to impede HEV infection. Another compound showing promise as an antiviral agent was the interferon receptor agonist RO8191 which reduces HEV replication and infection in hepatoma cell lines and in primary human hepatocytes.

As HEV is an RNA virus, prone to frequent mutations in its genome, it is likely that effective treatment of HEV infections will require combination therapies.

3.4. Risk Factors for Hepatitis E in Kidney Transplant Recipients (By PD Dr. Mira Choi)

Viral infections pose a significant challenge after kidney transplantation. Approximately 17% of the post‐transplant mortality in kidney transplant recipients was caused by infections [59]. Hepatitis is additionally among the top ten causes of mortality after a kidney transplantation [60]. Therefore, it is crucial to prevent an HEV infection in solid organ transplant (SOT) recipients. As SOT recipients receive immunosuppressive therapy to prevent transplant organ rejection, their risk for a chronic HEV infection is increased [61, 9]. In order to characterise HEV infections in kidney transplant recipients, Dr. Choi et al. analysed the course of chronic HEV infections in 17 kidney transplant recipients. They found that most of the HEV infections occur several months or even years after transplantation, when immunosuppressive therapy has been reduced. Additionally, they observed spontaneous remission in only one of the 17 patients and a higher risk for liver fibrosis. There were also signs of extrahepatic manifestations, indicated by the onset of proteinuria [62].

To identify risk factors of an HEV infection in kidney transplant recipients, Dr. Choi and her colleagues continued their research by conducting a study with 312 kidney transplant recipients with elevated liver enzymes, of which 18.9% were positive for HEV. They showed that most (78%) of the HEV‐infected individuals in their study were male and that there was no correlation with other comorbidities. Furthermore, the immunosuppressive regimen had an influence on the risk for chronic HEV infection. It was noticeable that 83% of HEV‐infected individuals were treated with tacrolimus, while only 5.1% received ciclosporin as calcineurin inhibitor treatment. It is important for kidney transplant recipients to avoid raw or undercooked meat or fish to prevent zoonotic infections with HEV. Dr. Choi also highlighted that meat and sausage products also need to be heated to over 70°C for at least 10 min to inactivate HEV. For several products like salami or ham, this might not be the case. Indeed, their study showed that kidney transplant recipients with an HEV infection consumed more pork products like pork steak, salami, or cured pork meat, and there were also more people who touched raw meat with their bare hands in the cohort with an HEV infection compared to the kidney transplant recipients without HEV [63]. These findings are supported by a study where commercially available pork products were tested for HEV RNA, of which 10% tested positive [64].

As a result, Dr. Choi concluded that immunosuppressive therapy after kidney transplantation provides a risk for a chronic HEV infection. In order to avoid such an infection, it is crucial to inform patients about the potential risk of pork products that have not been properly heated to be a source of infection. Hygiene measures during the handling of raw meat, like wearing gloves, should also be considered.

3.5. Round Table Discussion: Clinical Management of HEV—Challenge Accepted!

The panellists of the second round table discussed multiple challenges in the clinical management of HEV, which are summarised here in brief.