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To the Editor: The study by Rios-Muñoz et al. reporting rat hepatitis E virus (HEV) RNA in swine feces contains intriguing findings with the potential to change our understanding of rat HEV transmission routes (1). Of note, a substantial proportion of the positive samples in the study by Rios-Muñoz et al. exhibited high cycle threshold values, which might be suggestive of residual viral RNA. Field assessment of dog as sentinel animal for plague in endemic foci of Madagascar.

Hepatitis E virus (HEV) infection in humans is primarily caused by genotypes within Paslahepevirus species balayani (HEV-A). Rocahepevirus species ratti (HEV-C1, otherwise known as rat HEV) can also infect humans. HEV grows poorly in cell culture. Recent studies have reported that hyper-confluent cell layers, amphotericin B, MgCl2, progesterone, and dimethyl sulfoxide (DMSO) increase HEV yield in vitro. Here, we describe an independent evaluation of the effectiveness of these modifications in improving the yield of HEV-A genotype 4 (HEV-A4) and HEV-C1 from clinical samples in PLC/PRF/5 cells. We found that amphotericin B, MgCl2, and DMSO increased HEV yield from high-viral-load patient stool samples, while progesterone was not effective. Yield of HEV-C1 was lower than HEV-A4 across all medium conditions, but was boosted by DMSO. HEV-A4 could be maintained for over 18 months in amphotericin B- and MgCl2-containing medium, with the demonstration of viral antigen in supernatants and infected cells. We also evaluated various protocols to remove pseudo-envelopes from cell culture-derived HEV. Treating cell culture supernatant with NP-40 was the most effective. Our findings identify key modifications that boost HEV growth in vitro and illustrate the importance of independent verification of such studies using diverse HEV variants and cell lines.

Rat hepatitis E virus (rat HEV, Rocahepevirus genotype C1) represents a potential zoonotic threat, but its epidemiological and evolutionary characteristics in small mammals remain poorly understood, especially in regions with complex geography. Between 2022 and 2024, we collected 818 small mammals from seven border counties and cities in Yunnan, China. Rat HEV RNA was detected by RT-PCR, risk factors were assessed using binary logistic regression, and full genomes were sequenced for phylogenetic and molecular clock analysis. The overall prevalence of rat HEV was 6.23% (51/818), with significantly higher odds observed in Gengma and Heqing counties, in oriental house rat (Rattus tanezumi) and Chevrieri’s field mouse (Apodemus chevrieri), in residential habitats, and at mid-high altitudes (all p < 0.001). The 51 partial genomic sequences (RdRp gene) obtained in this study clustered within Rocahepevirus, forming two distinct subclades associated with host species. The two complete genomes, GS188 and GS197 from Rattus tanezumi, were classified as subtypes C1b and C1d, respectively. Bayesian analysis estimated that GS197 diverged from a closely related Rattus tanezumi-derived strain around 1998, while GS188 diverged from a lineage containing shrew and human strains around 1931. These findings reveal a relatively high prevalence and substantial genetic diversity of Rochepevirus in southwestern Yunnan, suggesting human-influenced transmission dynamics and a potential for cross-species infection.

Hepatitis E is a globally distributed human disease caused by hepatitis E virus (HEV). In Europe, it spreads through undercooked pork meat or other products and with blood components through transfusions. There are no approved or golden standard serologic systems for HEV diagnostics. Commercially available HEV tests often provide inconsistent results which may differ among the assays. In this study, we describe generation in yeast and characterization of HEV genotype 3 (HEV-3) and rat HEV capsid proteins self-assembled into virus-like particles (VLPs) and the development of HEV-specific monoclonal antibodies (MAbs). Full-length HEV-3 and rat HEV capsid proteins and their truncated variants comprising amino acids (aa) 112-608 were produced in yeast S. cerevisiae. The yeast-expressed rat HEV capsid protein was found to be glycosylated. The full-length HEV-3 capsid protein and both full-length and truncated rat HEV capsid proteins were capable to self-assemble into VLPs. All recombinant proteins contained HEV genotype-specific linear epitopes and cross-reactive conformational epitopes recognized by serum antibodies from HEV-infected reservoir animals. Two panels of MAbs against HEV-3 and rat HEV capsid proteins were generated. Their cross-reactivity pattern was investigated by Western blot, ELISA, and immunofluorescence assay on HEV-3-infected cell cultures. The analysis revealed cross-reactive, genotype-specific, and virus-reactive MAbs. MAb epitopes were localized within S, M, and P domains of HEV-3 and rat HEV capsid proteins. Yeast-generated recombinant VLPs of HEV-3 and rat HEV capsid proteins and HEV-specific MAbs might be employed to develop novel HEV detection systems.

Orthohepevirus C1, also known as rat hepatitis E virus (HEV), has been shown to sporadically cause disease in immunocompromised and immunocompetent adults. While routine serological assays vary in reactivity, rat HEV is not detected in routine HEV RT-PCR. Thus, such infections could be either missed or misclassified as conventional HEV (Orthohepevirus A) infections. We conducted a retrospective screening study among serum and plasma samples from patients suspected of having HEV infection, which were archived at the national consultant laboratory for HAV and HEV between 2000 and 2020. We randomly selected n = 200 samples, which were initially tested reactive (positive or borderline) for HEV-IgM and negative for HEV RNA and re-examined them using a highly sensitive Orthohepevirus C genotype 1-specific in-house RT-qPCR (LoD 95: 6.73 copies per reaction) and a nested RT-PCR broadly reactive for Orthohepevirus A and C. Conventional sanger sequencing was conducted for resulting PCR products. No atypical HEV strains were detected (0 of 200 [0.0%; 95% confidence interval: 0.0%–1.89%], indicating that Orthohepevirus C infections in the investigated population (persons with clinical suspicion of hepatitis E and positive HEV-IgM) are very rare.

The family Hepeviridae comprises the species Orthohepevirus A–D (HEV-A to -D). HEV-C genotype 1 (HEV-C1, rat HEV) is able to infect humans. This study investigated whether an optimized HEV-A cell culture system is able to propagate the cell culture-derived rat HEV, and if de novo isolation of the virus from rat liver is possible. We tested the liver carcinoma cell lines PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR for their susceptibility to HEV-C1 strains. Cells were infected with the cell culture-derived HEV-C1 strain R63 and rat liver-derived strain R68. Cells were maintained in MEMM medium, which was refreshed every 3–4 days. The viral load of HEV-C1 was determined by RT-qPCR in the supernatant and expressed as genome copies per mL (c/mL). Rat HEV replication was most efficient in the newly introduced HuH-7-Lunet BLR cell line. Even if the rat HEV isolate had been pre-adapted to PLC/PRF/5 by multiple passages, replication in HuH-7-Lunet BLR was still at least equally effective. Only HuH-7-Lunet BLR cells were susceptible to the isolation of HEV-C1 from the liver homogenate. These results suggest HuH-7-Lunet BLR as the most permissive cell line for rat HEV. Our HEV-C1 cell culture system may be useful for basic research, the animal-free generation of large amounts of the virus as well as for the testing of antiviral compounds and drugs.

The Asian musk shrew (shrew) is a new reservoir of a rat hepatitis E virus (HEV) that has been classified into genotype HEV-C1 in the species Orthohepevirus C. However, there is no information regarding classification of the new rat HEV based on the entire genome sequences, and it remains unclear whether rat HEV transmits from shrews to humans. We herein inoculated nude rats (Long-Evans rnu/rnu) with a serum sample from a shrew trapped in China, which was positive for rat HEV RNA, to isolate and characterize the rat HEV distributed in shrews. A rat HEV strain, S1129, was recovered from feces of the infected nude rat, indicating that rat HEV was capable of replicating in rats. S1129 adapted and grew well in PLC/PRF/5 cells, and the recovered virus (S1129c1) infected Wistar rats. The entire genomes of S1129 and S1129c1 contain four open reading frames and share 78.3–81.8% of the nucleotide sequence identities with known rat HEV isolates, demonstrating that rat HEVs are genetically diverse. We proposed that genotype HEV-C1 be further classified into subtypes HEV-C1a to HEV-C1d and that the S1129 strain circulating in the shrew belonged to the new subtype HEV-C1d. Further studies should focus on whether the S1129 strain infects humans.

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 (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.

Hepatitis E virus (HEV) is the chief cause of hepatitis E (inflammation of liver) across the globe. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF5. The additional reading frame encoded protein ORF5 protein's structure and function remain to be explored. Therefore, the presented study was conceptualized to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. The initial analysis revealed ORF5 protein as unstable, thermostable, hydrophilic and highly basic in nature. The primary structural analysis revealed higher percentages of amino acids Arg, Leu, Pro, Ser and Gly, which suggested that the ORF5 protein is richly endowed with some regulatory amino acids (Leu, Pro and Gly). The secondary structure of ORF5 protein showed all three major components (alpha-helix, beta-strand and random coil). The tertiary structure generated through homology modelling revealed mixed alpha/beta structural fold with subsequently higher percentage of strands and abundance of coils. Moreover, the surface analysis revealed the several clefts and tunnels along with few pores, clearly suggested the ability of ORF5 protein towards interaction with other molecules. The ORF5 protein was also identified with several post-translationally modified sites including glycosylation, phosphorylation and myriystoylation. The presence of these modified sites indicated the role of ORF5 protein in regulation. Thus, our analyses taken together interpret the ORF5 protein's essentiality in HEV. This data will help in exploring the prospective role of this additional genomic component of rat HEV through the sequence, structure and functional annotation of ORF5 protein.

Hepatitis E virus (HEV) of the family Hepeviridae, is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome is organized into six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF6 is attributed to life cycle of rat HEV. As ORF6 protein's remains to be explored in terms of its structural and functional implications, the following study was conceptualized to explore the prospective role of this additional genomic component of rat HEV. The detailed computational investigation was carried out for the ORF6 protein to elucidate its physiochemical properties, primary structure, secondary structure, tertiary structure and post-translational modifications, motif prediction and other functional characteristics. The in silico analysis revealed ORF6 protein as unstable, highly thermostable, hydrophobic and basic in nature. The amino acid compositional analysis revealed higher abundance of Leu, Arg, Ile and Pro amino acids in the polypeptide chain of ORF6 protein. The secondary structural analysis revealed all the three major elements, i.e., alpha-helices, beta-strands and coils. The generated 3D structural model of the ORF6 protein through homology modeling algorithm revealed mixed alpha/beta structural fold of the ORF6 protein with abundance of coils. Additionally, the structural models revealed the presence of clefts and a tunnel. The identified binding functions and the presence of several clefts suggested the commitment of ORF6 protein towards interaction with other ligand molecules. This theoretical study will facilitate towards deciphering the role of unexplored ORF6 encoded protein, thereby providing better understanding towards the pathogenesis of Norway rat HEVs.