Ancient evolutionary origins of hepatitis E virus in rodents

Hepatitis E virus (HEV; family Hepeviridae) infections cause >40,000 human deaths annually. Zoonotic infections predominantly originate from ungulates and occasionally from rats, highlighting the zoonotic potential of rodent-associated hepeviruses. We conducted host genomic data mining and uncovered two genetically divergent rodent-associated hepeviruses, and two bat-associated hepeviruses genetically related to known bat-associated strains. We thus analyzed 2,565 liver specimens from 108 rodent and shrew species sampled from globally understudied regions and hosts in Africa, Asia, and Latin America during 2011-2018 for hepeviruses by RT-PCR. We detected 63 positive field samples (2.5%, 95% CI 1.9-3.1) from 14 animal species, including two coinfections with genetically divergent strains and significant variation (X², P < 0.001) in detection rates between study sites. Strain-specific qRT-PCR assays showed virus concentrations between 9.2 × 10² and 3.2 × 10⁹ copies/g. We recovered 24 near-complete hepeviral genomes from rodents, shrews, and bats, all showing three partially overlapping open reading frames (ORFs), some including putative late domains that may be associated with quasi-envelopment. Rodent-derived hepeviruses grouped into five clades clustering in basal sister relationship to human- (31 to 84% distance in translated ORF1-3) and rat-associated HEV. Parsimony-based analyses and cophylogenetic reconciliations revealed that rodents were predominant sources of hepeviral cross-order host shifts. Bayesian ancestral state reconstructions substantiated a direct origin of human-associated HEV in ungulates such as swine and camelids (posterior probability 0.8), whereas the nonrecent evolutionary origins of human- and ungulate-associated HEV were projected to rodent hosts (posterior probability > 0.9). Our results elucidate the genealogy of human HEV and warrant increased surveillance and experimental risk assessments for rodent-associated hepeviruses.