Identification of novel rodent and shrew orthohepeviruses sheds light on hepatitis E virus evolution

The family Hepeviridae has seen an explosive expansion in its host range in recent years, yet the evolutionary trajectory of this zoonotic pathogen remains largely unknown. The emergence of rat hepatitis E virus (HEV) has introduced a new public health threat due to its potential for zoonotic transmission. This study investigated 2 464 wild small mammals spanning four animal orders, eight families, 21 genera, and 37 species in Yunnan Province, China. Using broadly reactive reverse transcription-polymerase chain reaction (RT-PCR), we systematically screened the presence and prevalence of Orthohepevirus and identified 192 positive specimens from 10 species, corresponding to an overall detection rate of 7.79%. Next-generation sequencing enabled the recovery of 24 full-length genomic sequences from eight host species, including Bandicota bengalensis, Eothenomys eleusis, and Episoriculus caudatus, representing newly reported host species for Orthohepevirus strains. Phylogenetic and sequence analyses revealed extensive genetic diversity within orthohepeviruses infecting rodents and shrews. Notably, among the identified strains, 20 were classified as Rocahepevirus ratti C1, two as C3, and one as Rocahepevirus eothenomi, while the remaining strain exhibited significant divergence, precluding classification. Evolutionary analyses highlighted close associations between orthohepeviruses and their respective host taxa, with distinct phylogenetic clustering patterns observed across different host orders. These findings emphasize the critical roles of co-speciation and cross-species transmission in shaping the evolutionary trajectories of the genera Paslahepevirus and Rocahepevirus.