Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes

Abstract

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Keywords: evolution; metagenomics; reverse genetics; virome; zoonosis.

Fig. 1.

Sampling sites and distribution of HBV-positive bat species. Sampling sites of HBV-positive bats are in red, and other sites are in yellow. Next to sites, the number of sampled species and specimens per family are given. Red, positive bat species; gray, distribution of positive bats.

Fig. 2.

Phylogenetic analysis including the unique bat viruses. (A) Bayesian phylogeny of Hepadnaviridae full genomes. The branch between ortho- and avihepadnaviruses was truncated for graphical reasons (interrupted lines). (B) ML full genome phylogeny. Unique bat viruses from this study are in red. Values at deep nodes represent Bayesian posterior probabilities or percentage ML bootstrap replicates; scale bars represent genetic distance.

Fig. 3.

Presentation of bat hepadnavirus infection. (A) Log10 hepadnavirus DNA copies per gram or milliliter. (B) H&E staining of hepadnavirus-positive hipposiderid bat GB09-301 liver. Arrow, lymphocytes; arrowheads, neutrophils and eosinophils. (C) In situ hybridization of hepadnavirus DNA in GB09-301 liver. (D) Immunofluorescence reaction pattern of hipposiderid bat serum GB09-256 with RBHBV-transfected HepG2 cells. (E) Reactivity of a polyclonal serum against the HBV core (green) and an mAb against an HBV surface epitope (red) in TBHBV-transfected HepG2 cells.

Fig. 4.

Inhibition of HBV infection by competing preS1 peptides. Newly synthesized and secreted HBsAg in supernatants of HBV-infected cultures 11–15 d postinfection of PHHs. Dashed red line, cutoff; dashed black line, IC₅₀ value.

Fig. 5.

Zoonotic potential of unique bat hepadnaviruses. (A) Infection with HDV_HBV or HDV_TBHBV. Presence (+) and absence (−) of specific myr-preS1 peptide inhibitors. ge, HDV genome equivalent 12 d postinfection. (B) Lack of protection by antisera from HB-vaccinated persons. C20/2 and HB1, mAbs against the HBV surface; IgG, nonspecific mAb; NS, serum of a non-HepB–vaccinated person. (C) HBV and TBHBV use hNTCP for infection. HepG2 cells expressing hNTCP (+) or a control (−) were incubated with HDV_HBV or HDV_TBHBV with (+) or without (−) inhibitors. *P < 0.05, **P < 0.02; t test. Cutoff, dashed red line.