Enhanced surveillance, subtyping, and host adaptation analysis reveal genotype-wide zoonotic potential of rat hepatitis E virus.

Background & Aims: Rocahepevirus ratti genotype 1 (rat hepatitis E virus; rHEV) is an emerging hepatitis agent that is genetically distinct from conventional human-infecting HEV. Zoonotic assessment of rHEV has been hampered by a lack of human-derived strain sequences and subtyping methods. We aimed to expand the dataset of human-derived rHEV genomes, develop a robust subtyping method, and investigate potential markers of human adaptation.

Methods: Human rHEV cases were detected in Hong Kong by population-wide PCR-based hepatitis surveillance. rHEV genomes were obtained using Sanger sequencing. New sequences were aligned with publicly available rHEV genomes to infer clade and subtype-level cut-offs based on maximum likelihood patristic distances. rHEV codon usage biases and selection pressures were measured, stratified by host, gene, and phylogenetic parameters. Bayesian phylogeny was used to estimate time to most recent common ancestor.

Results: Complete or partial rHEV sequences were obtained from 22 human rHEV cases identified in Hong Kong from 2017-2024. Distance-based subtyping of this enhanced dataset revealed two rHEV clades and six subtypes. Human-derived rHEV sequences clustered across both clades and four subtypes, indicating genotype-wide zoonotic potential. An open-source web application was created to classify sequences using this subtyping framework. Significantly lower codon usage bias was identified in ORF1 of human-derived rHEV strains. rHEV strains were well adapted to human codon usage patterns irrespective of the derived host. We observed significant selection relaxation in ORF2 of human-derived rHEV (k = 0.19; p = 5.48E-04), indicating higher tolerance to accumulation of non-synonymous mutations. The Hong Kong rHEV supercluster emerged c.30-50 years ago, decades before the first human case.

Conclusions: rHEV shows genotype-wide potential for human spillover infection across its phylogeny. Strengthened global surveillance and sequencing in both humans and rodents are essential to assess and manage this emerging threat.

Impact And Implications: Rat hepatitis E virus (rHEV) is an emerging cause of viral hepatitis in humans, but a comprehensive zoonotic assessment is hampered by a lack of sequences and systematic subtyping methods. Using a new, extended dataset of human-derived rHEV strains, we found a wide phylogenetic distribution of human-derived rHEV globally. Regardless of derived host, rHEV codon adaptation was similar for humans, indicating that all rHEV strains should be assumed to have zoonotic potential. Selection analyses revealed that human-derived sequences may be more tolerant to the accumulation of mutations at ORF2, which encodes the viral capsid. Some human-derived strains exhibited even lower codon usage bias, suggesting enhanced potential for replication and persistence across multiple hosts. These results are relevant to clinicians and policymakers as they prove the importance of instituting rHEV surveillance in humans and rodents.