Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and
musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human
hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV
surface proteins. Functional characterization of the shrew
sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface
peptide binding patterns, and
infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding
hepatitis B e antigen (
HBeAg) production and resembling those observed in
HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that
HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs.
HBeAg negativity is thus an ancient HBV
infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew
infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of
HBeAg-mediated HBV pathogenesis, entry, and species barriers.