The COVID-19 pandemic has demonstrated the significance of the human-animal interface in the emergence of zoonotic diseases, with wildlife serving as an important source of infection. A better understanding of the specific pathogens and mechanisms involved is vital to prepare against future outbreaks, especially in Southeast Asia, a hotspot for zoonotic diseases. This paper reviews the published literature on wildlife zoonoses in this region from 2012 to 2022. The results show a diverse range of potential zoonotic pathogens and the widespread occurrence of zoonotic diseases from wildlife. Drivers of zoonotic pathogen spillover include (i) environmental factors (e.g. animal habitat disruption, environmental conditions, exposure to contaminated water/food/soil), (ii) animal factors (e.g. movement patterns, age-related susceptibility), (iii) human factors (e.g. lack of awareness, poor hygiene practices, age, gender and income) and (iv) human-animal-environmental interface factors (e.g. close contact between humans and animals, exposure through visiting animals and presence of vectors). The diverse drivers of zoonoses in Southeast Asia put its communities at risk for infection. To mitigate these risks, global health efforts should consider adopting a One Health approach to foster collaboration across human, animal, and wildlife health sectors. This could involve educating communities on safe animal interactions and improving disease surveillance.

We previously observed a notable discrepancy in the distribution of HEV-3 subtypes between wastewater and clinical samples in Sweden. To confirm this observation and comprehensively elucidate HEV-3 circulation patterns across humans, animals, and environmental waters in Sweden, we analysed the HEV genetic diversity in archived wastewater samples between late 2016 and early 2018, clinical cases between 2012 and 2024, and all available Swedish sequences from the NCBI Virus database. HEV RNA was detected in all archived wastewater samples, with subtype 3c being the only subtype identified. In typed clinical cases, subtypes 3f (45/126) and 3c (44/126) were nearly equally distributed, though regional dominance varied. When incorporating human sequences from other Swedish studies, subtype 3f became dominant (75/168). Analysis of all available sequences revealed that 3f (113/136) was the dominant subtype in Sus scrofa (pigs and wild boars), while 3c (30/33) was dominant in environmental waters. These findings highlight the complex transmission dynamics of HEV-3 in Sweden. The near-absence of 3c in Swedish domestic pigs and wild boars, despite its high proportion in clinical cases, raises the question about the source of human 3c infection. In addition, the near-exclusive detection of 3c in wastewater suggests potential differences in viral shedding, disease severity of HEV-3 subtypes, or alternative host sources. This study emphasizes the importance of integrated One Health surveillance to track HEV circulation across reservoirs.

Time- and cost-saving surveillance of viral pathogens is achieved by tiled sequencing in which a viral genome is amplified in overlapping PCR amplicons and qPCR. However, designing pan-specific primers for viral pathogens with high genomic variability represents a significant challenge. Here, we present a bioinformatics command-line tool, called varVAMP (variable virus amplicons), which addresses this issue. It relies on multiple sequence alignments of highly variable virus sequences and enables degenerate primer design for qPCR or tiled amplicon whole genome sequencing. We demonstrate the utility of varVAMP by designing and evaluating novel pan-specific primer schemes suitable for sequencing the genomes of SARS-CoV-2, Hepatitis E virus, rat Hepatitis E virus, Hepatitis A virus, Borna-disease-virus-1, and Poliovirus using clinical samples. Importantly, we also designed primers on the same input data using the software packages PrimalScheme and Olivar and showed that varVAMP minimizes primer mismatches most efficiently. Finally, we established highly sensitive and specific Poliovirus qPCR assays that could potentially simplify current Poliovirus surveillance. varVAMP is open-source and available through PyPI, UseGalaxy, Bioconda, and https://github.com/jonas-fuchs/varVAMP .

Antibodies targeting the hepatitis E virus (HEV) surface capsid protein (CA) are essential for infection control and resolution, yet their molecular and functional attributes remain largely elusive. We characterized 144 human HEV-CA-specific monoclonal antibodies cloned from the memory B cells of HEV-exposed individuals. Most human anti-CA antibodies cross-reacted with all HEV genotype variants, and a subset also recognized the zoonotic rat hepatitis E virus. HEV antibody repertoire was diverse and contained highly potent neutralizing antibodies binding to the CA protruding (P) domain. Structural analyses of CA protein complexed with three potent and broad HEV antibodies uncovered a neutralizing site located on monomeric P domain loops at the apex of the viral spike. These findings provide valuable insights into the protective humoral response to HEV and offer a framework for the rational design of HEV vaccines and immunotherapies.

Hepatitis E virus (HEV) genotype 1 (HEV-1) infection in pregnant women is associated with adverse outcomes of pregnancy including fulminant hepatic failure, fetal loss, premature birth, and neonatal mortality, although the underlying mechanisms remain largely unclear. In this study, we first demonstrated that HEV-1 robustly infects pregnant gerbils and causes pregnancy-associated adverse outcomes, which were recorded in 4/6 HEV-1-infected but only 1/5 in PBS-inoculated pregnant gerbils. However, vertical transmission of HEV-1 from mothers to newborns is not evident, as HEV-1 RNA was not detected in uterus tissues or in newborn pups. To further determine whether HEV-1 can cross the placental barrier, we established an in vitro blood-placental barrier by coculturing human placental trophoblast cells (BeWo) and umbilical vein endothelial cells (HUVEC) in Transwell inserts. By using the placental barrier under the conditions in this study, we showed that quasi-enveloped or nonenveloped HEV-1, HEV-3, or HEV-4 virions do not readily cross the barrier prior to 4 d postinoculation when it has high barrier integrity. Importantly, we demonstrated that the placental barrier induces local antiviral resistance at the maternal-fetal interface, that interactions between maternal- and fetal-derived cocultured cells are important for induction of antiviral resistance, and that anti-HEV resistance can be transferred to nonplacental HepG2 liver cells. We also revealed that the main effectors of antiviral resistance at the placental barrier are type III interferons (IFN-λ1, λ2/3) and the chemokine CXCL10. The findings have important implications in understanding the mechanisms leading to HEV-1-associated maternal and fetal adverse outcomes in pregnant women.

Rocahepevirus ratti (rat hepatitis E virus; rHEV) is a ubiquitous pathogen of rats that has recently emerged as a cause of hepatitis in humans. Although several rHEV cases have been detected worldwide, the extent of human exposure to this hepatitis agent is still poorly defined. We conducted a multicenter surveillance study in China examining rHEV exposures in demographically diverse human populations from a One Health perspective.

In this multicenter cross-sectional study, we used fully validated rHEV IgG enzymatic immunoassays and reverse transcription PCR in 1,199 individuals with (n = 655) or without hepatitis (n = 544) recruited from three centers in China (Hainan, Hong Kong, and Shenzhen). The patient population included both urban and rural populations. Characteristics of infected individuals and phylogenetic links with rat epizootics were described.

rHEV IgG seroprevalence was higher in the rural Hainan cohort (15/229, 6.6%) compared with Shenzhen (9/427, 2.1%) and Hong Kong cohorts (2/543, 0.4%) (p <0.0001). This difference persisted on multivariable logistic regression with an adjusted odds ratio of 20.52 (95% CI: 13.86-30.39). rHEV exposure was also associated with increasing age and environmental rodent exposure. We observed rHEV viraemia in two hepatitis patients (2/655; 0.3%) in Hainan and Hong Kong with hepatitis B and renal transplantation, respectively. The latter developed chronic hepatitis E. 19/509 (3.7%) rats captured in Hainan harbored rHEV. Both human rHEV isolates were phylogenetically related to rodent-derived rHEV strains circulating in Hainan and Hong Kong, respectively.

Humans are intermittently exposed to rHEV, especially in rural settings with rodent exposure. Overt hepatitis occurs in individuals with liver disease or immunosuppression. Spillover strains are related to epizootics in rodents offering opportunities for targeted disinfestation.

Building on our previous work finding that Rocahepevirus ratti (rHEV) is a novel cause of sporadic viral hepatitis in humans, we studied rHEV exposures in humans from various epidemiological settings. We found intermittent exposure to rat hepatitis E in individuals living in both urban and rural settings with a markedly higher seroprevalence in the latter. Spillover viremic infections in individuals with underlying liver disease or immunosuppression were closely linked to epizootic rHEV strains circulating in rodents. Physicians and diagnostic laboratories should incorporate rHEV testing in algorithms for viral hepatitis while policymakers should consider rHEV surveillance in rodents to guide disinfestation efforts.

Hepatitis E virus (Paslahepevirus balayani [HEV]) is an important cause of acute viral hepatitis globally, with zoonotic genotypes linked to transmission through consumption of raw or undercooked swine meat or products. Recently, Rocahepevirus ratti (RHEV), member of Hepeviridae family, has emerged as a potential public health concern, with some human cases being reported. The present study aimed to investigate the presence of HEV, as well as RHEV in wastewaters from northern Portugal and Spain (nPS). Given the reported decline in HEV detection in swine from several regions of the world, we also aimed to explore HEV and RHEV in fattened swine fecal samples from the same region of the wastewaters. Between April 2020 and January 2022, a total of 44 wastewater samples were collected from wastewater treatment plants in nPS, alongside 400 fattened swine fecal samples from five farms of the same regions. Wastewater and swine fecal samples RNA extracts were screened for HEV using pangenotypic RT-qPCR and for RHEV using a RT-qPCR assay followed by characterization using nested RT-PCR. Regarding wastewaters, three tested positive for HEV, while 39 out of 44 tested positive for RHEV. Wastewater analysis in the Iberian Peninsula revealed a predominance of RHEV and a near absence of HEV. The absence of both viruses was observed in the swine fecal samples. This combined analysis showing near/total absence of HEV in wastewaters/fattened swine samples warrants further studies. High levels of RHEV in wastewater might also pose environmental transmission risks, particularly for individuals with occupational exposure, emphasizing the need for enhanced zoonotic virus surveillance in urban areas.

Viral hepatitis remains a global health challenge and immune status affects outcomes. In patients with haematological malignancies, including haematopoietic stem-cell transplantation recipients, viral hepatitis can be life-threatening due to the direct effects of the virus or the need to modify or delay chemotherapy. Additionally, haematopoietic stem-cell donors with past or current viral hepatitis infections might transmit the virus to recipients. The growing recognition of hepatitis E virus (HEV), advances in haematological therapies, and the availability of direct-acting antivirals for hepatitis C virus (HCV), led the 2022 9th European Conference on Infections in Leukaemia (ECIL-9) to update the 2013 ECIL-5 guidelines on viral hepatitis. The ECIL organising committee convened a panel of 13 impartial international experts (all authors of this Review) in viral hepatitis, both within and outside the fields of haematological malignancies and immunosuppression. The ECIL-9 panel conducted a review of the literature on hepatitis B virus (HBV), HCV, and HEV, grading the evidence based on the European Society for Clinical Microbiology and Infectious Diseases system. The panel identified key clinical questions and outcomes and built on the recommendations established during ECIL-5. A consensus conference was held in Sofia Antipolis, France, from Sept 15-17, 2022, bringing together 49 experts from 19 countries. The ECIL-9 panel presented the proposed recommendations, which were revised following expert discussions. A final consensus on updated guidelines was reached in a second plenary session. The updated ECIL-9 guidelines provide evidence-based recommendations on the prevention, screening, treatment, and long-term surveillance of viral hepatitis in patients with haematological malignancies and haematopoietic cell transplantation recipients.

The emergence of Rocahepevirus ratti genotype 1 (rat hepatitis E virus; rat HEV) in humans presents an unprecedented threat; however, the risk of rat HEV transmission to humans is not well understood. Here, we report the "Distinguishing Antibody Response Elicitation (DARE)" method, which distinguishes exposure to rat HEV. We use four study sets from China for large-scale population analysis: set 1 (hospital visit) and set 3 (ALT abnormality) from Yunnan province, a biodiversity hotspot, and set 2 (received physical examination) and set 4 (ALT abnormality) from Jiangsu province, a non-hotspot control region. rat HEV exposure risk is significantly higher in Yunnan, with 21.97% (190 of 865) in set 1 and 13.97% (70 of 501) in set 3, compared to 0.75% (9 of 1196) in Jiangsu's set 2. Six spillover infections for rat HEV are identified in set 1, with one case of abnormal ALT. The rat-1d strains carried by rats are closely related to those human infections. Our study reveals the substantial spillover burden posed by rat HEV in biodiversity hotspots and highlights the utility of DARE method for proactive surveillance of public health emergencies.

The Hepatitis E virus (HEV) is responsible for causing Hepatitis E, a zoonotic disease that has emerged as a significant global health concern, accounting for about 20 million infections and 70,000 deaths annually. Although it is often recognized as a disease that is acute in low-income countries, HEV has also been recognized as a zoonotic disease in high-income countries. The zoonotic transmission requires flexible approaches to effectively monitor the virus, vectors, and reservoirs. However, the environmental monitoring of HEV presents additional challenges due to limitations in current detection methods, making it difficult to accurately assess the global prevalence of the virus. These challenges hinder efforts to fully understand the scope of the disease and to implement effective control measures. This review will explore these and other critical concerns, addressing gaps in HEV research and highlighting the need for improved strategies in the monitoring, prevention, and management of Hepatitis E using a One Health approach.

Introduction: Hepatitis E virus (HEV) infection is caused by viruses belonging to the Hepeviridae family. HEV infection can be self-limiting; however, extrahepatic manifestations may be present. The purpose of the current study was to establish the seroprevalence of HEV among Bulgarian liver transplant recipients (LTRs) and to identify associated risk factors. Materials & Methods: The present study was conducted between April 1, 2023, and October 30, 2023, at the Military Medical Academy, Sofia, Bulgaria. All serum samples were tested for anti-HEV IgG/IgM using HEV IgG/IgM enzyme-linked immunosorbent assay on Dia.Pro (Milan, Italy). Each participating LTR completed a detailed paper-based closed-ended questionnaire regarding the associated risk factors for HEV infection. Results: The study included 73 LTRs with a mean age of 47.0 ± 14.0 years. Anti-HEV IgG antibodies were detected in 25 LTRs (34.2%), including 20 males (37.7%) and 5 females (25%). All participants were HEV-IgM negative. HEV seropositivity rates were higher but not statistically significant in LTRs aged >60 years than in those aged <60 years (40% vs. 32.7%). A significant factor by logistic regression was "high level of education" (odds ratio [OR] = 2.917; p = 0.038). Conclusion: To the best of our knowledge, this is the first seroepidemiological HEV study among LTRs from Bulgaria that found a high seroprevalence (34.2%).

Hepatitis E virus (HEV) is one of the most common pathogens causing acute hepatitis. Rat HEV, a member of the genus Rocahepevirus, infects mainly rat but can also cause human zoonotic infection. A survey of the virome of rats via next-generation sequencing (NGS) was performed in Yunnan Province and Inner Mongolia in China. Further screening of rat HEV was conducted by nested PCR. The complete genome of six representative strains were obtained by NGS and RT-PCR. The virome analysis revealed that multiple reads were annotated as Hepeviridae. The screening results showed that HEV was detected in 9.6% (34 of 355) of the rat samples and phylogenetically classified into three lineages. The sequences from Yunnan clustered with Rocahepevirus ratti, named the YnRHEV group, and those from Inner Mongolia were separated into two lineages, named the NmRHEV-1 and NmRHEV-2 groups. Complete sequence analysis showed that YnRHEV had very high sequence identity to a human HEV strain identified in immunosuppressed patients (88.7% to 94.3%), a reminder of the risk of cross-species transmission of rodent HEV. Notably, NmRHEV-1 and the most closely related rat HEV, RtCb-HEV/HeB2014, were divergent from other HEV. The phylogenetic analyses and lower sequence identities of the complete genome suggested the NmRHEV-1 to be a novel putative genus of the subfamily Orthohepevirinae. NmRHEV-2 shared the highest sequence identities (70.6% to 72.0%) with the species Rocahepevirus eothenomi, which may represent a putative novel genotype. This study revealed high genetic diversity of Hepeviridae in rats in China and a potentially zoonotic Rocahepevirus ratti strain.

Rat hepatitis E virus (ratHEV) is an emerging cause of acute hepatitis worldwide. The limited number of ratHEV cases may be associated with the lack of a proper molecular diagnosis method; thus, the clinical impact and breadth of ratHEV as a cause of acute hepatitis remain uncertain.

The study was carried out in four phases. I) Identification: Molecular assays were identified through a literature search. II) Testing: The methods were evaluated in a rodent testing cohort, and the most suitable molecular diagnosis algorithm was established. III) Derivation: The established algorithm was tested in a larger rodent cohort. IV) Clinical validation: The algorithm was used in a cohort of individuals suffering from acute hepatitis of unknown aetiology, thus establishing the frequency of ratHEV as an aetiological agent of acute hepatitis and its clinical impact.

We detected differences in the frequency of positive results among the assays evaluated. After comparing all available molecular methods, we established a molecular diagnostic algorithm, which revealed that 17.5% of the 103 rodents in the validation cohort were infected with ratHEV. In the clinical validation cohort, of 562 patients with acute hepatitis of unknown origin, 8 cases of ratHEV infection were identified during the three years of the study, representing a frequency of 1.4%. One (37.5%) case had severe acute hepatitis; four (50.0%) patients required hospitalization, one of whom (12.5%) died. The strains detected in these patients revealed a close phylogenetic relationship with those found in rats in Spain.

Our study demonstrated that ratHEV is an emerging and underdiagnosed cause of acute hepatitis. The results provide evidence that ratHEV should be monitored and included in the differential diagnosis of acute hepatitis.

ClinicalTrials.gov Identifier: NCT05062967 IMPACT AND IMPLICATIONS: While rat hepatitis E virus (ratHEV) is a newly emerging zoonotic virus worldwide, the results of the present study indicate that the molecular diagnosis methods for this virus may be inappropriate. After establishing a proper molecular diagnostic algorithm using available assays, we demonstrated that ratHEV is an emerging and underdiagnosed aetiological agent of acute hepatitis of unknown origin. The results also expand the knowledge of the diversity of ratHEV strains capable of infecting humans in Europe. These findings strongly suggest that ratHEV should be monitored and included in the differential diagnosis of acute hepatitis.

Hepatitis E virus (HEV) causes 3.3 million symptomatic cases and 44,000 deaths per year. Chronic infections can arise in immunocompromised individuals, and pregnant women may suffer from fulminant disease as a consequence of HEV infection. Despite these important implications for public health, no specific antiviral treatment has been approved to date. Here, we report combined functional, biochemical, and X-ray crystallographic studies that characterize the human antibody response in convalescent HEV patients. We identified a class of potent and broadly neutralizing human antibodies (bnAbs), targeting a quaternary epitope located at the tip of the HEV capsid protein pORF2 that contains an N-glycosylation motif and is conserved across members of the Hepeviridae. These glycan-sensitive bnAbs specifically recognize the non-glycosylated pORF2 present in infectious particles but not the secreted glycosylated form acting as antibody decoy. Our most potent bnAb protects human liver-chimeric mice from intraperitoneal HEV challenge and co-housing exposure. These results provide insights into the bnAb response to this important emerging pathogen and support the development of glycan-sensitive antibodies to combat HEV infection.

Clinical and experimental studies have advanced our understanding of hepatitis E virus (HEV) infection; however, translating the findings to improve prevention and clinical outcomes remains challenging. Phylogenetic analyses of HEV show inconsistencies due to variations in the nucleotide regions studied. This study examined specific HEV regions to facilitate comprehensive molecular and phylogenetic analyses by examining the complete genome and commonly studied partial genome regions. We compared topological similarities between phylogenetic trees and evaluated evolutionary divergence using base substitutions and pairwise distances. The hypervariable region (HVR) showed the closest topology (Robinson-Foulds, Jaccard Robinson-Foulds and clustering information) to the complete genome and a higher mutation rate, resulting in longer branch lengths and clearer genotypic distinctions. Pairwise analysis revealed greater intra- and intergenotypic diversity in the HVR than in other regions. The higher base substitution rate and longer branch lengths of HVR suggest its key role in genotype evolution. Classifying HEV using HVR instead of the other partial genomic regions can reveal subtypes that more accurately reflect the genetic characteristics of HEV. Future research could focus on HVRs to better compare clinical symptoms and genetic features of HEV.

Hepatitis E virus (HEV) causes sporadic cases in industrialized countries and endemic outbreaks in areas with lower sanitation standards. The wide host reservoir of HEV makes it a potential source of new zoonotic transmission and dissemination in humans. Thus, the perception of HEV as a confined ailment has shifted to one of global concern. Considering HEV's environmental stability and heterogeneity in the host range of HEV's genotypes, various transmission pathways and sources for HEV infections are plausible. Here, we provide an overview on HEV's transmission routes and discuss the role of HEV as a foodborne zoonosis, as well as preventive measures and open research questions.

Zoonotic hepatitis E virus (HEV) infection is an emerging global public health concern, and understanding the dynamics of HEV transmission between animals and humans is crucial for public health. Animal models are critical to advancing the understanding of HEV pathogenesis, drug screening, vaccine development, and other related areas. Here, we provide an overview of recent studies investigating the cross-species transmission of HEV, and also delve into the current research and application of animal HEV infection models including non-human primates, rodents, pigs, and chickens, offering a comprehensive assessment of the advantages and disadvantages of each model. This review highlights the findings related to viral replication, shedding patterns, and immune response in these animal models, and discusses the implications for our understanding of HEV transmission to humans. These advancements in the field enhance our understanding of the biological traits and pathogenic mechanisms of HEV, offering robust support for the development of highly effective and targeted prevention and treatment strategies.

Hepatitis E virus (HEV) is considered as an emerging zoonotic pathogen circulating in a wide range of animals. In recent decades, the genus Paslahepevirus frequently isolated in pigs were the most involved in human clinical practice. In addition, the genus Rocahepevirus have been isolated in rodents, and transmission to humans is increasingly reported worldwide, although gaps remain regarding the exposure factors. In this study, the presence of HEV was investigated in urban wastewater, swine slaughterhouse wastewater and river waters, in a geographical area where its circulation had previously been reported. In addition to the expected detection of Paslahepevirus in almost all waters samples collected, Rocahepevirus strains were detected with the same frequencies in urban and river waters, at concentrations up to 40-fold higher. No Rocahepeviruses were detected in swine slaughterhouse wastewater. This is the first study demonstrating the presence of Rocahepevirus in French wastewater. Although no evidence of transmission was reported among patients followed for a suspected HEV infection in the same area between April 2019 and October 2023 (i.e. 135/3078 serological tests positive for anti-HEV IgM detection; 46/822 blood samples positive for Paslahepevirus genome detection but none for Rocahepevirus), the circulation of Rocahepevirus in waters in such concentrations raises the question of the possible zoonotic transmission to human. Indeed, the waterborne transmission of HEV is now well documented in industrialized countries, and the exploration of the growing number of human infections in Europe involving Rocahepevirus has not until now made it possible to clarify the transmission routes.

Hepatitis E virus (HEV) variants infecting humans belong to two species: Paslahepevirus balayani (bHEV) and Rocahepevirus ratti (rat hepatitis E virus; rHEV). R. ratti is a ubiquitous rodent pathogen that has recently been recognized to cause hepatitis in humans. Transmission routes of rHEV from rats to humans are currently unknown. In this study, we examined rHEV exposure in cats and dogs to determine if they are potential reservoirs of this emerging human pathogen. Virus-like particle-based IgG enzymatic immunoassays (EIAs) capable of differentiating rHEV & bHEV antibody profiles and rHEV-specific real-time RT-PCR assays were used for this purpose. The EIAs could detect bHEV and rHEV patient-derived IgG spiked in dog and cat sera. Sera from 751 companion dogs and 130 companion cats in Hong Kong were tested with these IgG enzymatic immunoassays (EIAs). Overall, 13/751 (1.7%) dogs and 5/130 (3.8%) cats were sero-reactive to HEV. 9/751 (1.2%) dogs and 2/130 (1.5%) cats tested positive for rHEV IgG, which was further confirmed by rHEV immunoblots. Most rHEV-seropositive animals were from areas in or adjacent to districts reporting human rHEV infection. Neither 881 companion animals nor 652 stray animals carried rHEV RNA in serum or rectal swabs. Therefore, we could not confirm a role for cats and dogs in transmitting rHEV to humans. Further work is required to understand the reasons for low-level seropositivity in these animals.

Hepatitis E virus (HEV) and Rat Hepatitis E virus (RHEV), recognized for their zoonotic potential, pose significant public health concerns. Our previous research identified both viruses in effluent wastewater in Gothenburg, Sweden. However, there are lingering inquiries regarding the prevalence and genetic diversity of these viruses in influent wastewater, as well as the utility of wastewater surveillance in elucidating their community circulation dynamics. To address these knowledge gaps, we conducted weekly collection of wastewater samples at the Rya wastewater treatment plant in Gothenburg throughout 2023. The concentrations of HEV and RHEV were quantified using quantitative polymerase chain reaction (qPCR). Additionally, two semi/nested-PCR were utilized to amplify viral strains. Furthermore, HEV strains from patients within the same region, as well as other regions in Sweden in 2023, were incorporated into the analysis. Remarkably, we observed a high prevalence of HEV (86%) and RHEV (98%) in wastewater samples, with the majority of HEV sequences identified as subtype 3c/i (9/12). In contrast, HEV subtype 3f was the most sequenced among clinical patient samples (6/12). Notably, previously unreported HEV-3b and unclassified strains were detected in wastewater. Almost all RHEV strains (20/21) were clustered into European groups, with none of the RHEV genetically close to strains previously found in human cases. The notable discordance in prevalence and identified subtypes of HEV-3 in wastewater compared to clinical samples suggests either a significant underdiagnosis of HEV infections or differences in viral loads and shedding durations among humans between HEV-3 subtypes. This underscores the urgent need for improved diagnostic techniques and heightened awareness of HEV transmission dynamics. Furthermore, the consistent detection of RHEV in wastewater underscores the necessity for further investigations to assess the potential role of RHEV in hepatitis cases of unknown etiology, given that most currently available clinical diagnostic assays fail to detect RHEV.

Circovirus human is a new viral species that includes the human circovirus (HCirV), which has been linked to hepatitis in immunocompromised persons. We investigated prevalence of HCirV infection in 278 patients with hepatitis and 184 asymptomatic persons using real-time PCR and sequencing assays. HCirV viremia and sequences were found in 8 (2.9%) hepatitis patients and no asymptomatic patients. Alternate causes of hepatitis (hepatitis E and cholangitis) were clearly identifiable in 2 HCirV-infected patients. HCirV could not be ruled out as a contributor to hepatitis in the remaining 6 patients, 4 of whom were immunocompromised. Persistent infections were documented in 3 patients, but only 1 had relapsing hepatitis. One HCirV patient displayed symptoms of an infectious mononucleosis-like syndrome. Isolates clustered with known HCirV strains from France and China. HCirV-derived virus-like particles bound to PLC/PRF/5 and Hep-G2 human hepatoma cells but not to lung epithelial cells, indicating hepatic tropism.

Rat hepatitis E virus (ratHEV; species Rocahepevirus ratti) is considered a newly emerging cause of acute hepatitis of zoonotic origin. ratHEV infection of people living with HIV (PLWH) might portend a worse, as with hepatitis E virus (HEV; species Paslahepevirus balayani), and consequently this group may constitute a high-risk population. We aimed to evaluate the prevalence of ratHEV by measuring viral RNA and specific IgG antibodies in a large Spanish cohort of PLWH. Multicentre study conducted in Spain evaluating PLWHIV included in the Spanish AIDS Research Network (CoRIS). Patients were evaluated for ratHEV infection using PCR at baseline and anti-ratHEV IgG by dot blot analysis to evaluate exposure to ratHEV strains. Patients with detectable ratHEV RNA were followed-up to evaluate persistence of viremia and IgG seroconversion. Eight-hundred and forty-two individuals were tested. A total of 9 individuals showed specific IgG antibodies against ratHEV, supposing a prevalence of 1.1 (95% CI; 0.5%-2.1%). Of these, only one was reactive to HEV IgG antibodies by ELISA. One sample was positive for ratHEV RNA (prevalence of infection: 0.1%; 95% CI: 0.08%-0.7%). The case was a man who had sex with men exhibiting a slightly increased alanine transaminase level (49 IU/L) as only biochemical alteration. In the follow-up, the patients showed undetectable ratHEV RNA and seroconversion to specific ratHEV IgG antibodies. Our study shows that ratHEV is geographical broadly distributed in Spain, representing a potential zoonotic threat.

Leptospirosis and hantavirus syndrome are two major rodent-borne diseases in Taiwan. Rocahepevirus ratii (RHEV), a virus closely related to hepatitis E virus (HEV, Paslahepevirus balayani), is emerging and has been reported to cause hepatitis in humans. We employed wastewater-based epidemiology to actively monitor rodent-borne pathogens, and the correlations with human cases were evaluated. Wastewater was collected using grab sampling at 11 sites along a sewer system including influents and effluents at a wastewater treatment plant in Tamsui, New Taipei City, Taiwan, monthly during June 2023 to May 2024. The presence of pathogens was examined by reverse transcription-polymerase chain reaction (RT-PCR). The result showed an overall positivity rate of 38.2% (50/131). Leptospira was detected most often (48/131, 36.6%), and RHEV and hantaviruses were found once each during the study period. Sequencing identified Leptospira interrogans close to isolates from rodents and human cases, while sequences of hantavirus and RHEV were most similar to isolates from rodents. No significant correlation was found with human cases or positive samples for rodent DNA. Here, we present an example of a One Health approach applying wastewater to environmental surveillance for the early detection and prevention of emerging diseases.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

Classically, all hepatitis E virus (HEV) variants causing human infection belong to the genus Paslahepevirus (HEV-A). However, the increasing cases of rat HEV infection in humans since 2018 challenged this dogma, posing increasing health threats. Herein, we investigated the underlying mechanisms dictating the zoonotic potentials of different HEV species and their possible cross-protection relationships. We found that rat HEV virus-like particles (HEVVLPs) bound to human liver and intestinal cells/tissues with high efficiency. Moreover, rat HEVVLPs and infectious rat HEV particles penetrated the cell membrane and entered human target cells postbinding. In contrast, ferret HEVVLPs showed marginal cell binding and entry ability, bat HEVVLPs and avian HEVVLPs exhibited no binding and entry potency. Structure-based three-dimensional mapping identified that the surface spike domain of rat HEV is crucial for cell binding. Antigenic cartography indicated that rat HEV exhibited partial cross-reaction with HEV-A. Intriguingly, sera of HEV-A infected patients or human HEV vaccine Hecolin® immunized individuals provided partial cross-protection against the binding of rat HEVVLPs to human target cells. In summary, the interactions between the viral capsid and cellular receptor(s) regulate the distinct zoonotic potentials of different HEV species. The systematic characterization of antigenic cartography and serological cross-reactivity of different HEV species provide valuable insights for the development of species-specific diagnosis and protective vaccines against zoonotic HEV infection.

Since Mongolian gerbils are broadly susceptible to hepatitis E virus (HEV), including genotypes 1, 4, 5, and 8 (HEV-1, HEV-5, HEV-5, and HEV-8) and rat HEV, they are a useful small animal model for HEV. However, we have observed that the subtypes HEV-3k and HEV-3ra in genotype 3 HEV (HEV-3) were not infected efficiently in the gerbils. A small-animal model for HEV-3 is also needed since HEV-3 is responsible for major zoonotic HEV infections. To investigate whether gerbils can be used as animal models for other subtypes of HEV-3, we injected gerbils with five HEV-3 subtypes (HEV-3b, -3e, -3f, -3k, and -3ra) and compared the infectivity of the subtypes. We detected viral RNA in the gerbils' feces. High titers of anti-HEV IgG antibodies in serum were induced in all HEV-3b/ch-, HEV-3f-, and HEV-3e-injected gerbils. Especially, the HEV-3e-injected animals released high levels of viruses into their feces for an extended period. The virus replication was limited in the HEV-3b/wb-injected and HEV-3k-injected groups. Although viral RNA was detected in HEV-3ra-injected gerbils, the copy numbers in fecal specimens were low; no antibodies were detected in the sera. These results indicate that although HEV-3's infectivity in gerbils depends on the subtype and strain, Mongolian gerbils have potential as a small-animal model for HEV-3. A further comparison of HEV-3e with different genotype strains (HEV-4i and HEV-5) and different genera (rat HEV) revealed different ALT elevations among the strains, and liver damage occurred in HEV-4i- and HEV-5-infected but not HEV-3e- or rat HEV-infected gerbils, demonstrating variable pathogenicity across HEVs from different genera and genotypes in Mongolian gerbils. HEV-4i- and HEV-5-infected Mongolian gerbils might be candidate animal models to examine HEV's pathogenicity.

Two of 11 children with acute hepatitis of unknown origin were found to have rat hepatitis E virus infection. This infection should be considered in the differential diagnosis of children with acute hepatitis of unknown origin.

Previous studies have emphasized the necessity of surveillance and control measures for hepatitis E virus (HEV) infection in wild boars, an important reservoir of HEV. To assess the current situation of HEV infection in wild boars in Japan, this study investigated the prevalence and genetic diversity of HEV among wild boars captured in 16 prefectures of Japan during 2018-2023. Serum samples from 968 wild boars were examined for anti-HEV IgG antibodies and HEV RNA. The prevalence of anti-HEV IgG varied geographically from 0 % to 35.0 %. HEV RNA was detected in 3.6 % of boars, with prevalence varying by prefecture from 0 % to 22.2 %. Genotype 3 was the most prevalent genotype (91.9 %), followed by genotype 4 (5.4 %), with one strain closely related to genotype 6. The prevalence of HEV infection among wild boars decreased from 2018/2019 to 2022/2023 with significant declines in levels of anti-HEV IgG antibodies (14.5 % vs. 6.2 %, P < 0.0001) and HEV RNA (7.6 % vs. 1.5 %, P < 0.0001). Regional analysis showed varying trends, with no HEV RNA-positive boars found in several regions in recent years. A plausible factor contributing to the decline in HEV infection is the application of countermeasures, including installing fences to prevent intrusion into pig farms, implemented in response to the emergence of classical swine fever virus (CSFV) infection in wild boars and domestic pigs, with incidents reported annually since 2018. Further investigation is warranted to explore the association between countermeasures to CSFV infection and the decrease in HEV infection among wild boars.